Oxidation of methionine residues: the missing link between stress and signalling responses in plants.

PubMed

Emes, Michael J

2009-08-13

In response to biotic and abiotic stresses, plants induce a complex array of pathways and protein phosphorylation cascades which generally lead to a response aimed at mitigating the particular insult. In many cases, H2O2 has been implicated as the signalling molecule, but, although progress has been made in assembling the downstream components of these signalling pathways, far less is known about the mechanism by which the signal is perceived. In this issue of the Biochemical Journal, Hardin et al. provide evidence for a plausible mechanism by which plants perceive H2O2. Evidence is presented for chemical oxidation of methionine residues by H2O2 at critical hydrophobic positions within the canonical motifs that define the phosphorylation sites of a number of enzymes, thus inhibiting binding of protein kinases. This process is reversible by MSR (methionine sulfoxide reductase) activity in vivo. Using synthetic peptides for a number of enzymes which are phosphorylated by families of protein kinases, including the CDPK (calcium-dependent protein kinase) and AMPK (AMP-activated protein kinase) families, coupled with in vivo studies of assimilatory plant nitrate reductase, the authors demonstrate that this mechanism regulates the ability of kinases to bind the target protein, directly linking oxidative signals to changes in protein phosphorylation. These results may have widespread implications for the perception of redox signalling in plants and animals.

Altered Skeletal Muscle Mitochondrial Proteome As the Basis of Disruption of Mitochondrial Function in Diabetic Mice

PubMed Central

Zabielski, Piotr; Lanza, Ian R.; Gopala, Srinivas; Holtz Heppelmann, Carrie J.; Bergen, H. Robert; Dasari, Surendra

2016-01-01

Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and β-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of β-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a β-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress. PMID:26718503

Enhancement of nitrite on heme-induced oxidative reactions: A potential toxicological implication.

PubMed

Lu, Naihao; Chen, Wei; Zhu, Jingjie; Peng, Yi-Yuan

2012-02-01

Evidence to support the role of heme as major inducers of oxidative damage is increasingly present. Nitrite (NO(2)(-)) is one of the major end products of NO metabolism. Although the biological significance of heme/NO(2)(-)-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO(2)(-) on heme-dependent redox reaction have been greatly underestimated. In this study, we investigated the influence of NO(2)(-) on heme -dependent oxidative reactions. It was found that NO(2)(-) had the capacity to act as a reducing agent to remove high oxidation states of heme iron. In the reduction of ferryl heme to ferric heme, NO(2)(-) was oxidized to a nitrating agent NO(2), and subsequently, tyrosine residues in bovine serum albumin (BSA) were nitrated. However, the presence of NO(2)(-) surprisingly exerted pro-oxidant effect on heme-H(2)O(2)-induced formation of BSA carbonyls at lower concentrations and enhanced the loss of HepG2 cell viability dose-dependently, which was probably due to the ability of this inorganic compound to efficiently enhance the peroxidase activity and oxidative degradation of heme. These data provide novel evidence that the dietary intake and experimental use of NO(2)(-) in vivo and in vitro would possess the pro-oxidant activity through interfering in heme-dependent oxidative reactions. Besides the classic role in protein tyrosine nitration, the deleterious effects on heme redox reactions may provide new insights into the toxicological implications of NO(2)(-) with cellular heme proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Prolonged Fasting Identifies Heat Shock Protein 10 as a Sirtuin 3 Substrate

PubMed Central

Lu, Zhongping; Chen, Yong; Aponte, Angel M.; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N.

2015-01-01

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

Oxidative Stress, Amyloid-β Peptide, and Altered Key Molecular Pathways in the Pathogenesis and Progression of Alzheimer’s Disease

PubMed Central

Butterfield, D. Allan; Boyd-Kimball, Debra

2018-01-01

Oxidative stress is implicated in the pathogenesis and progression of Alzheimer’s disease (AD) and its earlier stage, amnestic mild cognitive impairment (aMCI). One source of oxidative stress in AD and aMCI brains is that associated with amyloid-β peptide, Aβ1-42 oligomers. Our laboratory first showed in AD elevated oxidative stress occurred in brain regions rich in Aβ1-42, but not in Aβ1-42-poor regions, and was among the first to demonstrate Aβ peptides led to lipid peroxidation (indexed by HNE) in AD and aMCI brains. Oxidatively modified proteins have decreased function and contribute to damaged key biochemical and metabolic pathways in which these proteins normally play a role. Identification of oxidatively modified brain proteins by the methods of redox proteomics was pioneered in the Butterfield laboratory. Four recurring altered pathways secondary to oxidative damage in brain from persons with AD, aMCI, or Down syndrome with AD are interrelated and contribute to neuronal death. This “Quadrilateral of Neuronal Death” includes altered: glucose metabolism, mTOR activation, proteostasis network, and protein phosphorylation. Some of these pathways are altered even in brains of persons with preclinical AD. We opine that targeting these pathways pharmacologically and with lifestyle changes potentially may provide strategies to slow or perhaps one day, prevent, progression or development of this devastating dementing disorder. This invited review outlines both in vitro and in vivo studies from the Butterfield laboratory related to Aβ1-42 and AD and discusses the importance and implications of some of the major achievements of the Butterfield laboratory in AD research. PMID:29562527

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance

PubMed Central

Simberg, Dmitri; Park, Ji-Ho; Karmali, Priya P.; Zhang, Wan-Ming; Merkulov, Sergei; McCrae, Keith; Bhatia, Sangeeta; Sailor, Michael; Ruoslahti, Erkki

2009-01-01

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles. PMID:19394687

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance.

PubMed

Simberg, Dmitri; Park, Ji-Ho; Karmali, Priya P; Zhang, Wan-Ming; Merkulov, Sergei; McCrae, Keith; Bhatia, Sangeeta N; Sailor, Michael; Ruoslahti, Erkki

2009-08-01

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles.

N-Acetylcysteine treatment of dystrophic mdx mice results in protein thiol modifications and inhibition of exercise induced myofibre necrosis.

PubMed

Terrill, Jessica R; Radley-Crabb, Hannah G; Grounds, Miranda D; Arthur, Peter G

2012-05-01

Oxidative stress is implicated as a factor that increases necrosis of skeletal muscles in Duchenne Muscular Dystrophy (DMD) and the dystrophic mdx mouse. Consequently, drugs that minimize oxidative stress are potential treatments for muscular dystrophy. This study examined the in vivo benefits to mdx mice of an antioxidant treatment with the cysteine precursor N-acetylcysteine (NAC), administered in drinking water. NAC was completely effective in preventing treadmill exercise-induced myofibre necrosis (assessed histologically) and the increased blood creatine kinase levels (a measure of sarcolemma leakiness) following exercise were significantly lower in the NAC treated mice. While NAC had no effect on malondialdehyde level or protein carbonylation (two indicators of irreversible oxidative damage), treatment with NAC for one week significantly decreased the oxidation of glutathione and protein thiols, and enhanced muscle protein thiol content. These data provide in vivo evidence for protective benefits of NAC treatment on dystropathology, potentially via protein thiol modifications. Copyright © 2011 Elsevier B.V. All rights reserved.

Protein Mediated Oxidative Stress in Patients with Diabetes and its Associated Neuropathy: Correlation with Protein Carbonylation and Disease Activity Markers

PubMed Central

Almogbel, Ebtehal

2017-01-01

Introduction Free radicals have been implicated as Diabetes Mellitus (DM) contributors in type 2 DM and its associated Diabetes Mellitus Neuropathy (DMN). However, the potential for protein mediated oxidative stress to contribute disease pathogenesis remains largely unexplored. Aim To investigate the status and contribution of protein mediated oxidative stress in patients with DM or DMN and to explore whether oxidative protein modification has a role in DM progression to DM associated neuropathy. Materials and Methods Sera from 42 DM and 37 DMN patients with varying levels of disease activities biomarkers (HbA1C, patients’ age or disease duration) and 21 age- and sex-matched healthy controls were evaluated for serum levels of protein mediated oxidative stress. Results Serum analysis showed significantly higher levels of protein carbonyl contents in both DM and DMN patients compared with healthy controls. Importantly, not only was there an increased number of subjects positive for protein carbonylation, but also the levels of protein carbonyl contents were significantly higher among DM and DMN patients, whose HbA1C were ≥8.8 as compared with patients with lower HbA1C (HbA1C<8.8). Similar pattern of protein carbonyls formation was also observed with patients’ ages or with patient’s disease durations, suggesting a possible relationship between protein oxidation and disease progression. Furthermore, sera from DMN patients had higher levels of protein carbonylation compared with non-neuropathic DM patients’ sera, suggesting an involvement of protein oxidation in the progression of diabetes to diabetes neuropathy. Conclusion These findings support an association between protein oxidation and DM or DMN progression. The stronger response observed in patients with higher HbA1C or patients’ ages or disease durations suggests, that protein mediated oxidative stress may be useful in evaluating the progression of DM and its associated DMN and in elucidating the mechanisms of these disorders pathogenesis. PMID:28384853

Glycation, oxidation and glycoxidation of IgG: a biophysical, biochemical, immunological and hematological study.

PubMed

Islam, Sidra; Moinuddin; Mir, Abdul Rouf; Raghav, Alok; Habib, Safia; Alam, Khursheed; Ali, Asif

2017-09-12

Glycation and oxidation induce structural alterations in the proteins in an interdependent manner with consequent pathological implications. The published literature presents wide range of modifications in conformational characteristics of proteins by glycation and oxidation; however, there is little data that could elaborate the cumulative effect of both the processes. This study has analysed the modifications in IgG by methylglyoxal (MG) (glycative stress), hydroxyl radical ([Formula: see text]) (oxidative stress) and by their combined action i.e. [Formula: see text] treatment of MG glycated IgG (glycoxidation). It further addresses the implications of the altered structural integrity of IgG on its immunological characteristics and impact on haematological parameters in rabbits. Using circular dichroism, FTIR, SDS-PAGE analysis, thioflavin-T fluorescence assay, congo red absorbance analysis, dynamic light scattering, transmission electron microscopy, ELISA, blood cell counts and rectal temperature studies, we report that the glycoxidative modification caused maximum alteration in the IgG as compared to the glycatively and oxidatively modified protein. Far-UV CD results confirmed the highest decline in the beta-pleated sheet content of the protein by glycoxidation. The damage led to the reduced flexibility and enhanced electronic interactions in IgG as observed by near-UV CD. Modifications caused cross-linking and adduct formation in the serum protein. The electron micrograph confirmed amorphous aggregation in modified IgG. The modifications increased the hydrodynamic radius of IgG by allowing the attachment of [Formula: see text] and MG residues. The glycoxidatively modified IgG induced the maximum antibody titres that showed high specificity towards the altered IgG. The glycoxidation of IgG leads to activation of inflammatory pathways.

Lighting the fat furnace without SFRP5

PubMed Central

Rauch, Alexander; Mandrup, Susanne

2012-01-01

WNT signaling plays a central role in the regulation of cellular growth and differentiation. In this issue of the JCI, Mori et al. link WNT signaling to the oxidative capacity of adipocytes during obesity. They show that secreted frizzled-related protein 5 is an extracellular matrix–residing protein that is highly induced during obesity and inhibits oxidative phosphorylation in a tissue-autonomous manner, possibly by sequestering WNT3a. These results implicate local WNT signaling as an attractive target for combating obesity. PMID:22728932

Average oxidation state of carbon in proteins

PubMed Central

Dick, Jeffrey M.

2014-01-01

The formal oxidation state of carbon atoms in organic molecules depends on the covalent structure. In proteins, the average oxidation state of carbon (ZC) can be calculated as an elemental ratio from the chemical formula. To investigate oxidation–reduction (redox) patterns, groups of proteins from different subcellular locations and phylogenetic groups were selected for comparison. Extracellular proteins of yeast have a relatively high oxidation state of carbon, corresponding with oxidizing conditions outside of the cell. However, an inverse relationship between ZC and redox potential occurs between the endoplasmic reticulum and cytoplasm. This trend provides support for the hypothesis that protein transport and turnover are ultimately coupled to the maintenance of different glutathione redox potentials in subcellular compartments. There are broad changes in ZC in whole-genome protein compositions in microbes from different environments, and in Rubisco homologues, lower ZC tends to occur in organisms with higher optimal growth temperature. Energetic costs calculated from thermodynamic models are consistent with the notion that thermophilic organisms exhibit molecular adaptation to not only high temperature but also the reducing nature of many hydrothermal fluids. Further characterization of the material requirements of protein metabolism in terms of the chemical conditions of cells and environments may help to reveal other linkages among biochemical processes with implications for changes on evolutionary time scales. PMID:25165594

A FLUORIMETRIC SEMI-MICROPLATE FORMAT ASSAY OF PROTEIN CARBONYLS IN BLOOD PLASMA

PubMed Central

Mohanty, Joy G.; Bhamidipaty, Surya; Evans, Michele K.; Rifkind, Joseph M.

2010-01-01

Oxidative stress, originating from reactive oxygen species (ROS), has been implicated in aging and various human diseases. The ROS generated can oxidize proteins producing protein carbonyl derivatives. The level of protein carbonyls in blood plasma has been used as a measure of overall oxidative stress in the body. Classically, protein carbonyls have been quantitated spectrophotometrically by directly reacting them with 2,4, dinitrophenylhydrazine (DNPH). However, the applicability of this method to biological samples is limited by its low inherent sensitivity. This limitation has been overcome by the development of sensitive ELISA methods to measure protein carbonyls. As part of the Healthy Aging in Neighborhoods of Diversity across the Lifespan study, oxidative stress in humans were quantified by measuring blood plasma protein carbonyls using the two commercially available ELISA kits and the spectrophotometric DNPH assay. Surprisingly, two ELISA methods gave very different values for protein carbonyls that were both different from the spectrophotometric method. We have developed a fluorescent semi-microplate format assay of protein carbonyls involving direct reaction of protein carbonyls with fluorescein thiosemicarbazide that correlates (R=0.992) with the direct spectrophotometric method. It has a coefficient of variation of 4.99% and is at least 100 times more sensitive than the spectrophotometric method. PMID:20122892

Oxidative Risk for Atherothrombotic Cardiovascular Disease

PubMed Central

Leopold, Jane A.; Loscalzo, Joseph

2009-01-01

In the vasculature, reactive oxidant species including reactive oxygen, nitrogen, or halogenating species, and thiyl, tyrosyl, or protein radicals, may oxidatively modify lipids and proteins with deleterious consequences for vascular function. These biologically active free radical and non-radical species may be produced by increased activation of oxidant-generating sources and/or decreased cellular antioxidant capacity. Once formed, these species may engage in reactions to yield more potent oxidants that promote transition of the homeostatic vascular phenotype to a pathobiological state that is permissive for atherothrombogenesis. This dysfunctional vasculature is characterized by lipid peroxidation and aberrant lipid deposition, inflammation, immune cell activation, platelet activation, thrombus formation, and disturbed hemodynamic flow. Each of these pathobiological states is associated with an increase in the vascular burden of free radical species-derived oxidation products and, thereby, implicates increased oxidant stress in the pathogenesis of atherothrombotic vascular disease. PMID:19751821

Characterization of oxidation end product of plasma albumin 'in vivo'.

PubMed

Musante, Luca; Bruschi, Maurizio; Candiano, Giovanni; Petretto, Andrea; Dimasi, Nazzareno; Del Boccio, Piero; Urbani, Andrea; Rialdi, Giovanni; Ghiggeri, Gian Marco

2006-10-20

Anti-oxidants are paradoxically much lower in plasma than inside cells even blood is comparably exposed to the oxidative stress. 'In vitro' models suggest a critical role of albumin as substitutive anti-oxidant in plasma but no proof for this role is available 'in vivo.' Herein, we demonstrate by LC/MS/MS that plasma albumin undergoes massive oxidation in primary nephrotic syndrome, involving stable sulphonation SO3- of the free SH of Cys 34 with +48Da increase in exact mass of the protein (ESI-MS) and formation of a fast moving isoform in the pH range between 5 and 7. Physical-chemical experiments with DSC and fluorescence spectra indicate a thermal stabilization of the structure upon oxidation. This is the first demonstration of massive oxidation of albumin 'in vivo' that reflects a functional role of the protein. Free radicals should be implicated in the pathogenesis of proteinuria in human FSGS.

Protective activity of green tea against free radical- and glucose-mediated protein damage.

PubMed

Nakagawa, Takako; Yokozawa, Takako; Terasawa, Katsutoshi; Shu, Seiji; Juneja, Lekh Raj

2002-04-10

Protein oxidation and glycation are posttranslational modifications that are implicated in the pathological development of many age-related disease processes. This study investigated the effects of green tea extract, and a green tea tannin mixture and its components, on protein damage induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (a free radical generator) and glucose in in vitro assay systems. We found that green tea extract can effectively protect against protein damage, and showed that its action is mainly due to tannin. In addition, it was shown that the chemical structures of tannin components are also involved in this activity, suggesting that the presence of the gallate group at the 3 position plays the most important role in the protective activity against protein oxidation and glycation, and that there is also a contribution by the hydroxyl group at the 5' position in the B ring and the sterical structure. These findings demonstrate the mechanisms of the usefulness of green tea in protein oxidation- and glycation-associated diseases.

Ascorbate attenuates pulmonary emphysema by inhibiting tobacco smoke and Rtp801-triggered lung protein modification and proteolysis.

PubMed

Gupta, Indranil; Ganguly, Souradipta; Rozanas, Christine R; Stuehr, Dennis J; Panda, Koustubh

2016-07-19

Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxido-nitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage.

Ascorbate attenuates pulmonary emphysema by inhibiting tobacco smoke and Rtp801-triggered lung protein modification and proteolysis

PubMed Central

Gupta, Indranil; Ganguly, Souradipta; Rozanas, Christine R.; Stuehr, Dennis J.

2016-01-01

Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung. Using a guinea pig model and human lung cells, we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of lung injury, but also activate Rtp801, a key proinflammatory cellular factor involved in tobacco smoke-induced lung damage. Rtp801 triggers nuclear factor κB and consequent inducible NOS (iNOS)-mediated overproduction of NO, which in combination with excess superoxide produced during Rtp801 activation, contribute to increased oxido-nitrosative stress and lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts lung protein nitration but fails to prevent or reverse the major tobacco smoke-induced oxidative lung injury. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent such damage by inhibiting both tobacco smoke-induced lung protein oxidation as well as activation of pulmonary Rtp801 and consequent iNOS/NO-induced nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, resulting in emphysematous lung damage. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco smoke-induced emphysema. Overall, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the noncellular and cellular damage mechanisms governing emphysematous lung injury and demonstrate the potential of vitamin C to accomplish holistic prevention of such damage. PMID:27382160

Dynamic Redox Regulation of IL-4 Signaling.

PubMed

Dwivedi, Gaurav; Gran, Margaret A; Bagchi, Pritha; Kemp, Melissa L

2015-11-01

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation.

Dynamic Redox Regulation of IL-4 Signaling

PubMed Central

Dwivedi, Gaurav; Gran, Margaret A.; Bagchi, Pritha; Kemp, Melissa L.

2015-01-01

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation. PMID:26562652

The effect of oxidation on the mechanical response and microstructure of porcine aortas.

PubMed

Stephen, Elizabeth A; Venkatasubramaniam, Arundhathi; Good, Theresa A; Topoleski, L D Timmie

2014-09-01

Reactive oxygen species (ROS), a product of many cellular functions, has been implicated in many age-related pathophysiological processes, including cardiovascular disease. The arterial proteins collagen and elastin may also undergo structural and functional changes due to damage caused by ROS. This study examined the effect of oxidation on the mechanical response of porcine aortas and aorta elastin and the associated changes in structural protein ultrastructure as a step in exploring the role of molecular changes in structural proteins with aging on elastic artery function. We examined the change in mechanical properties of aorta samples after various oxidation times as a first step in understanding how the oxidative environment associated with aging could impact mechanical properties of arterial structural proteins. We used confocal microscopy to visualize how the microstructure of isolated elastin changed with oxidation. We find that short term oxidation of elastin isolated from aortas leads to an increase in material stiffness, but also an increase in the fiber diameter, increase in void space in the matrix, and a decrease in the fiber orientation, possibly due to fiber cross-linking. The short term effects of oxidation on arterial collagen is more complex, with increase in material stiffness seen in the collagen region of the stress stretch curve at low extents of oxidation, but not at high levels of oxidation. These results may provide insight into the relationship between oxidative damage to tissue associated with aging and disease, structure of the arterial proteins elastin and collagen, and arterial mechanical properties and function. © 2013 Wiley Periodicals, Inc.

Interference of flavonoids with fluorescent intracellular probes: methodological implications in the evaluation of the oxidative burst by flow cytometry.

PubMed

Peluso, Ilaria; Manafikhi, Husseen; Reggi, Raffaella; Palmery, Maura

2014-08-01

The evaluation of oxidative burst is particularly relevant in many pathological and subclinical conditions. Flow cytometry provides quick and accurate measures of the reactive oxygen species production by leukocytes in most situations. However, spurious results, related to probes' efflux may be observed in several instances. Many factors affect the evaluation of the oxidative burst with fluorescent probes that require intracellular deacetylation and could be substrate of the multidrug resistance proteins (MDR). After discussing the implications of the efflux of fluorophores in the normalization strategies in flow cytometry assays, we have pointed out the possible interference of flavonoids with fluorescet probes' staining and signal. We have also reviewed the results from human intervention studies regarding the evaluation of oxidative burst with these probes. In vitro, at concentrations close to post-ingestion circulating levels, some flavonoids and their metabolites could interfere with probes' staining and fluorescence signal through different mechanisms, such as the inhibition of esterases, the modulation of the MDR-mediate efflux of probe and the inhibition of the oxidation of probe. These effects may explain the contrasting results obtained by human intervention studies. Finally, also inflammatory state or the use of drugs substrate of MDR proteins could affect the evaluation of the oxidative burst with intracellular probes. © 2014 International Society for Advancement of Cytometry.

A universal entropy-driven mechanism for thioredoxin–target recognition

PubMed Central

Palde, Prakash B.; Carroll, Kate S.

2015-01-01

Cysteine residues in cytosolic proteins are maintained in their reduced state, but can undergo oxidation owing to posttranslational modification during redox signaling or under conditions of oxidative stress. In large part, the reduction of oxidized protein cysteines is mediated by a small 12-kDa thiol oxidoreductase, thioredoxin (Trx). Trx provides reducing equivalents for central metabolic enzymes and is implicated in redox regulation of a wide number of target proteins, including transcription factors. Despite its importance in cellular redox homeostasis, the precise mechanism by which Trx recognizes target proteins, especially in the absence of any apparent signature binding sequence or motif, remains unknown. Knowledge of the forces associated with the molecular recognition that governs Trx–protein interactions is fundamental to our understanding of target specificity. To gain insight into Trx–target recognition, we have thermodynamically characterized the noncovalent interactions between Trx and target proteins before S-S reduction using isothermal titration calorimetry (ITC). Our findings indicate that Trx recognizes the oxidized form of its target proteins with exquisite selectivity, compared with their reduced counterparts. Furthermore, we show that recognition is dependent on the conformational restriction inherent to oxidized targets. Significantly, the thermodynamic signatures for multiple Trx targets reveal favorable entropic contributions as the major recognition force dictating these protein–protein interactions. Taken together, our data afford significant new insight into the molecular forces responsible for Trx–target recognition and should aid the design of new strategies for thiol oxidoreductase inhibition. PMID:26080424

Hemin-Graphene Derivatives with Increased Peroxidase Activities Restrain Protein Tyrosine Nitration.

PubMed

Xu, Huan; Yang, Zhen; Li, Hailing; Gao, Zhonghong

2017-12-14

Protein tyrosine nitration is implicated in the occurrence and progression of pathological conditions involving free radical reactions. It is well recognized that hemin can catalyze protein tyrosine nitration in the presence of nitrite and hydrogen peroxide. Generally, the catalytic efficiency is positively correlated to its peroxidase activity. In this study, however, it is found that the efficiency of hemin in catalyzing protein tyrosine nitration is largely suppressed after functionalization with graphene derivatives, even though its peroxidase-like activity is more than quadrupled. Further studies show that the oxidation of tyrosine is still observed for these composites; dityrosine formation, however, is greatly inhibited. Furthermore, these composites also exhibit strong effects on the oxidation of nitrite into nitrate. Therefore, we propose a mechanism in which hemin-graphene derivatives facilitate the oxidation of tyrosine and nitrite to produce tyrosyl radicals and nitrogen dioxide radicals in the presence of hydrogen peroxide, but graphene interlayers serve as barriers that hinder radical-radical coupling reactions; consequently, protein tyrosine nitration is restrained. This property of hemin-graphene derivatives, by which they catalyze substrate oxidation but suppress radical-radical coupling reactions, shows their great potential in selective oxidation procedures for byproduct removal. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Vitamin C mediates chemical aging of lens crystallins by the Maillard reaction in a humanized mouse model

PubMed Central

Fan, Xingjun; Reneker, Lixing W.; Obrenovich, Mark E.; Strauch, Christopher; Cheng, Rongzhu; Jarvis, Simon M.; Ortwerth, Beryl J.; Monnier, Vincent M.

2006-01-01

Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. We hypothesized that the Maillard reaction, which leads browning and aroma development during the baking of foods, would occur between the lens proteins and the highly reactive oxidation products of vitamin C. To test this hypothesis, we engineered a mouse that selectively overexpresses the human vitamin C transporter SVCT2 in the lens. Consequently, lenticular levels of vitamin C and its oxidation products were 5- to 15-fold elevated, resulting in a highly compressed aging process and accelerated formation of several protein-bound advanced Maillard reaction products identical with those of aging human lens proteins. These data strongly implicate vitamin C in lens crystallin aging and may serve as a model for protein aging in other tissues particularly rich in vitamin C, such as the hippocampal neurons and the adrenal gland. The hSVCT2 mouse is expected to facilitate the search for drugs that inhibit damage by vitamin C oxidation products. PMID:17075057

Effects of quercetin on hemoglobin-dependent redox reactions: relationship to iron-overload rat liver injury.

PubMed

Lu, Nai-Hao; Chen, Chao; He, Ying-Jie; Tian, Rong; Xiao, Qiang; Peng, Yi-Yuan

2013-01-01

Flavonoids have been widely reported to protect liver injury in iron-overload diseases, where the mechanism of this therapeutic action is dependent on their antioxidant effects, including free radical scavenging and metal-chelating. In this study, in contrast to the significant decrease in iron content, quercetin (Qu) from lower diet (0.3%, w/w) showed pro-oxidant ability on protein carbonyl formation and exhibited unobvious effect on iron-overload rat liver injury. Furthermore, the anti- and pro-oxidant activities of Qu on hemoglobin (Hb)-dependent redox reactions (i.e. the oxidative stability of Hb and its cytotoxic ferryl intermediate, Hb-induced protein oxidation) were investigated to illustrate the elevated protein oxidation in lower Qu-treated iron-overload rat. It was found that superoxide (O₂·⁻) and hydrogen peroxide (H₂O₂) were generated during the reaction between Qu and Hb. Qu, however, effectively reduced ferryl intermediate back to ferric Hb in a biphasic kinetic reaction. Moreover, Qu could significantly aggravate Hb-H₂O₂-induced protein oxidation at low concentrations and exhibit protective effects at high concentrations. Different from the classic antioxidant mechanisms of Qu, the dual effects on Hb redox reactions in vitro, therefore, may provide new insights into the physiological and pharmacological implications of Qu with iron-overload disease.

Effect of long term chilled (up to 5 weeks) then frozen (up to 12 months) storage at two different sub-zero holding temperatures on beef: 3. Protein structure degradation and a marker of protein oxidation.

PubMed

Holman, Benjamin W B; Coombs, Cassius E O; Morris, Stephen; Kerr, Matthew J; Hopkins, David L

2018-05-01

Different chilled (~0.1 °C for up to 5 weeks) then frozen storage (up to 12 months) combinations and two frozen storage holding temperatures (-12 °C and -18 °C) effects on beef M. longissimus lumborum (LL) protein structure degradation and a marker of protein oxidation were tested. Particle size (PS) analysis and protein solubility results found storage combination effects on protein degradation to be significant (P < 0.05), although the influence of frozen holding temperatures was negligible. LL carbonyl, and nitrate and nitrite content responses were variable and yet broadly reflected an increased incidence of protein oxidation across increasing chilled storage and ensuing frozen storage periods - this aspect meriting future exploration. Total myoglobin content and the estimated myoglobin redox fractions (metmyoglobin, deoxymyoglobin, and oxymyoglobin) were also subject to storage treatment. These findings demonstrate the capacity for beef storage selection (chilled-then-frozen) to manage compositional protein changes and its implications on sensory quality traits across comparative 'long term' durations. Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.

The dual effects of nitrite on hemoglobin-dependent redox reactions.

PubMed

Lu, Naihao; Chen, Chao; He, Yingjie; Tian, Rong; Xiao, Qiang; Peng, Yi-Yuan

2014-08-31

Evidence to support the role of heme proteins-dependent reactions as major inducers of oxidative damage is increasingly present. Nitrite (NO2(-)) is one of the major end products of NO metabolism, and from the daily consumption. Although the biological significance of heme proteins/NO2(-)-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO2(-) on heme proteins-dependent redox reactions have been greatly underestimated. In this study, we investigated the influence of NO2(-) on met-hemoglobin (Hb)-dependent oxidative and nitrative stress. It was found that NO2(-) effectively reduced cytotoxic ferryl intermediate back to ferric Hb in a biphasic kinetic reaction. However, the presence of NO2(-) surprisingly exerted pro-oxidant effect on Hb-H2O2-induced protein (bovine serum albumin, enolase) oxidation at low concentrations and enhanced the loss of HepG2 cell viability. In the reduction of ferryl Hb to ferric state, NO2(-) was decreased and oxidized to a nitrating agent NO2, Tyr12 and Tyr191 in enolase were subsequently nitrated. In contrast to the frequently inhibitive effect of nitrotyrosine, NO2(-)-triggered tyrosine nitration might play an important role in enolase activation. These data provided novel evidence that the dietary intake and potential therapeutic application of NO2(-) would possess anti- and pro-oxidant activities through interfering in hemoglobin-dependent redox reactions. Besides the classic role in protein tyrosine nitration, the dual effects on hemoglobin-triggered oxidative stress may provide new insights into the physiological and toxicological implications of NO2(-) with heme proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Formoterol attenuates increased oxidative stress and myosin protein loss in respiratory and limb muscles of cancer cachectic rats

PubMed Central

Salazar-Degracia, Anna; Busquets, Sílvia; Argilés, Josep M.; López-Soriano, Francisco J.

2017-01-01

Muscle mass loss and wasting are characteristic features of patients with chronic conditions including cancer. Therapeutic options are still scarce. We hypothesized that cachexia-induced muscle oxidative stress may be attenuated in response to treatment with beta2-adrenoceptor-selective agonist formoterol in rats. In diaphragm and gastrocnemius of tumor-bearing rats (108 AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with formoterol (0.3 mg/kg body weight/day for seven days, daily subcutaneous injection), redox balance (protein oxidation and nitration and antioxidants) and muscle proteins (1-dimensional immunoblots), carbonylated proteins (2-dimensional immunoblots), inflammatory cells (immunohistochemistry), and mitochondrial respiratory chain (MRC) complex activities were explored. In the gastrocnemius, but not the diaphragm, of cancer cachectic rats compared to the controls, protein oxidation and nitration levels were increased, several functional and structural proteins were carbonylated, and in both study muscles, myosin content was reduced, inflammatory cell counts were greater, while no significant differences were seen in MRC complex activities (I, II, and IV). Treatment of cachectic rats with formoterol attenuated all the events in both respiratory and limb muscles. In this in vivo model of cancer-cachectic rats, the diaphragm is more resistant to oxidative stress. Formoterol treatment attenuated the rise in oxidative stress in the limb muscles, inflammatory cell infiltration, and the loss of myosin content seen in both study muscles, whereas no effects were observed in the MRC complex activities. These findings have therapeutic implications as they demonstrate beneficial effects of the beta2 agonist through decreased protein oxidation and inflammation in cachectic muscles, especially the gastrocnemius. PMID:29255650

Oxidative Stress in Spinocerebellar Ataxia Type 7 Is Associated with Disease Severity.

PubMed

Torres-Ramos, Y; Montoya-Estrada, A; Cisneros, B; Tercero-Pérez, K; León-Reyes, G; Leyva-García, N; Hernández-Hernández, Oscar; Magaña, Jonathan J

2018-06-06

Spinocerebellar ataxia type 7 is a neurodegenerative inherited disease caused by a CAG expansion in the coding region of the ATXN7 gene, which results in the synthesis of polyglutamine-containing ataxin-7. Expression of mutant ataxin-7 disturbs different cell processes, including transcriptional regulation, protein conformation and clearance, autophagy, and glutamate transport; however, mechanisms underlying neurodegeneration in SCA7 are still unknown. Implication of oxidative stress in the pathogenesis of various neurodegenerative diseases, including polyglutamine disorders, has recently emerged. We perform a cross-sectional study to determine for the first time pheripheral levels of different oxidative stress markers in 29 SCA7 patients and 28 age- and sex-matched healthy subjects. Patients with SCA7 exhibit oxidative damage to lipids (high levels of lipid hydroperoxides and malondialdehyde) and proteins (elevated levels of advanced oxidation protein products and protein carbonyls). Furthermore, SCA7 patients showed enhanced activity of various anti-oxidant enzymes (glutathione reductase, glutathione peroxidase, and paraoxonase) as well as increased total anti-oxidant capacity, which suggest that activation of the antioxidant defense system might occur to counteract oxidant damage. Strikingly, we found positive correlation between some altered oxidative stress markers and disease severity, as determined by different clinical scales, with early-onset patients showing a more severe disturbance of the redox system than adult-onset patients. In summay, our results suggest that oxidative stress might contribute to SCA7 pathogenesis. Furthermore, oxidative stress biomarkers that were found relevant to SCA7 in this study could be useful to follow disease progression and monitor therapeutic intervention.

ATM directs DNA damage responses and proteostasis via genetically separable pathways

PubMed Central

Lee, Ji-Hoon; Mand, Michael R.; Kao, Chung-Hsuan; Zhou, Yi; Ryu, Seung W.; Richards, Alicia L.; Coon, Joshua J.; Paull, Tanya T.

2018-01-01

The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATM causes Ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. Here, we genetically separated DNA damage activation of ATM from oxidative activation using separation-of-function mutations. We found that deficiency in ATM activation by Mre11-Rad50-Nbs1 and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATM had minimal effects on DNA damage-related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of ATM lacking oxidative activation generates widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATM activation and its effects on cellular metabolism and DNA damage responses in human cells and implicates ATM in the control of protein homeostasis. PMID:29317520

Fatiguing contractions increase protein S-glutathionylation occupancy in mouse skeletal muscle

DOE PAGES

Kramer, Philip A.; Duan, Jicheng; Gaffrey, Matthew J.; ...

2018-05-23

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 minmore » after the last stimulation and processed for redox proteomics assay of S-glutathionylation.« less

Fatiguing contractions increase protein S-glutathionylation occupancy in mouse skeletal muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kramer, Philip A.; Duan, Jicheng; Gaffrey, Matthew J.

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 minmore » after the last stimulation and processed for redox proteomics assay of S-glutathionylation.« less

Triage of oxidation-prone proteins by Sqstm1/p62 within the mitochondria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Minjung; Shin, Jaekyoon, E-mail: jkshin@med.skku.ac.kr

2011-09-16

Highlights: {yields} The mitochondrion contains its own protein quality control system. {yields} p62 localizes within the mitochondria and forms mega-dalton sized complexes. {yields} p62 interacts with oxidation-prone proteins and the proteins of quality control. {yields} In vitro delivery of p62 improves mitochondrial functions. {yields} p62 is implicated as a participant in mitochondrial protein quality control. -- Abstract: As the mitochondrion is vulnerable to oxidative stress, cells have evolved several strategies to maintain mitochondrial integrity, including mitochondrial protein quality control mechanisms and autophagic removal of damaged mitochondria. Involvement of an autophagy adaptor, Sqstm1/p62, in the latter process has been recently described.more » In the present study, we provide evidence that a portion of p62 directly localizes within the mitochondria and supports stable electron transport by forming heterogeneous protein complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of mitochondrial proteins co-purified with p62 revealed that p62 interacts with several oxidation-prone proteins, including a few components of the electron transport chain complexes, as well as multiple chaperone molecules and redox regulatory enzymes. Accordingly, p62-deficient mitochondria exhibited compromised electron transport, and the compromised function was partially restored by in vitro delivery of p62. These results suggest that p62 plays an additional role in maintaining mitochondrial integrity at the vicinity of target machineries through its function in relation to protein quality control.« less

Unraveling the role of animal heme peroxidases in superoxide mediated Mn oxide formation

NASA Astrophysics Data System (ADS)

Learman, D. R.; Hansel, C. M.

2013-12-01

Manganese(III,IV) oxides are important in the environment as they can impact the fate of a broad range of nutrients (e.g. carbon and phosphate) and contaminates (e.g. lead and chromium). Bacteria play a valuable role in the production of Mn oxides, yet the mechanisms and physiological reasons remain unclear. Roseobacter sp. AzwK-3b, an organism within the abundant and ubiquitous Roseobacter clade, has recently been shown to oxidize Mn(II) via a novel pathway that involves enzymatic extracellular superoxide production. However, in reactions with only Mn(II) and abiotically generated superoxide, we find superoxide alone is not enough to produce Mn(III,IV) oxides. Scavenging of the byproduct hydrogen peroxide (via the addition of catalase) is required to generate Mn oxides via abiotic reaction of Mn(II) with superoxide. Thus, R. AzwK-3b must produce superoxide and also scavenge hydrogen peroxide to form Mn oxides. Further, in-gel Mn(II) oxidation assay revealed a protein band that could generate Mn oxides in the presence of soluble Mn(II). This Mn(II)-oxidizing protein band was excised from the gel and the peptides identified via mass spectrometry. An animal heme peroxidase (AHP) was the predominant protein found in this band. This protein is homologous to the AHPs previously implicated as a Mn(II)-oxidizing enzyme within the Alphaproteobacteria, Erythrobacter SD-21 and Aurantimonas manganoxydans strain SI85-9A1. Currently, protein expression of the AHPs in R. AzwK-3b is being examined to determine if expression is correlated with Mn(II) concentration or oxidative stress. Our data suggests that AHPs do not directly oxidize Mn(II) but rather plays a role in scavenging hydrogen peroxide and/or producing an organic Mn(III) ligand that complexes Mn(III) and likely aids in Mn oxide precipitation.

Carvedilol: relation between antioxidant activity and inhibition of the mitochondrial permeability transition.

PubMed

Oliveira, Paulo J; Esteves, Telma; Rolo, Anabela P; Monteiro, Pedro; Gonçalves, Lino; Palmeira, Carlos M; Moreno, António J

2003-01-01

The mitochondrial permeability transition (MPT) is an event related to severe oxidative stress (for example, during myocardial ischemia and reperfusion) and excessive mitochondrial calcium accumulation, also being implicated in cell death. In this study, we compared the effect of carvedilol on the cardiac MPT induced by calcium and phosphate (Ca/Pi) and calcium/carboxyatractyloside (Ca/Catr). Oxidative stress plays a major role in MPT induction by Ca/Pi, leading to the oxidation of protein thiol groups, in contrast with Ca/Catr, where such oxidation is secondary to MPT induction and is not caused by oxidative stress. Mitochondria were isolated from rat hearts and parameters related to MPT induction were evaluated (n = 5 for each inducer): mitochondrial swelling and oxidation of protein thiol groups (both measured by spectrophotometry). Using Ca/Pi, carvedilol protected mitochondria from MPT induction, particularly in its high conductance form. Its effect was demonstrated by analyzing the decrease in mitochondrial swelling amplitude. Simultaneously, we observed inhibition of protein thiol group oxidation (p < 0.001). By contrast, carvedilol did not show any protective effect with Ca/Catr. Carvedilol was only effective against the MPT when the oxidation of protein thiol groups was the cause and not the consequence of the MPT phenomenon. The results clearly show that during myocardial aggressions (ischemia and reperfusion, for example), the protective effect of carvedilol is primarily due to an antioxidant mechanism, inhibiting the production and effects of reactive oxygen species.

A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

PubMed Central

Piek, Susannah; Kahler, Charlene M.

2012-01-01

The Gram-negative bacterial cell envelope consists of an inner membrane (IM) that surrounds the cytoplasm and an asymmetrical outer-membrane (OM) that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS), phospholipids, outer membrane proteins (OMPs), and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella, and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation, and isomerization pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, the pathways required for the biosynthesis of the OM and the regulatory circuits that control them have evolved to suit the lifestyle of each organism. PMID:23267440

Vascular Nox (NADPH Oxidase) Compartmentalization, Protein Hyperoxidation, and Endoplasmic Reticulum Stress Response in Hypertension.

PubMed

Camargo, Livia L; Harvey, Adam P; Rios, Francisco J; Tsiropoulou, Sofia; Da Silva, Renée de Nazaré Oliveira; Cao, Zhenbo; Graham, Delyth; McMaster, Claire; Burchmore, Richard J; Hartley, Richard C; Bulleid, Neil; Montezano, Augusto C; Touyz, Rhian M

2018-07-01

Vascular Nox (NADPH oxidase)-derived reactive oxygen species and endoplasmic reticulum (ER) stress have been implicated in hypertension. However, relationships between these processes are unclear. We hypothesized that Nox isoforms localize in a subcellular compartment-specific manner, contributing to oxidative and ER stress, which influence the oxidative proteome and vascular function in hypertension. Nox compartmentalization (cell fractionation), O 2 - (lucigenin), H 2 O 2 (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase, peroxiredoxin oxidation), and ER stress (PERK [protein kinase RNA-like endoplasmic reticulum kinase], IRE1α [inositol-requiring enzyme 1], and phosphorylation/oxidation) were studied in spontaneously hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). VSMC proliferation was measured by fluorescence-activated cell sorting, and vascular reactivity assessed in stroke-prone SHR arteries by myography. Noxs were downregulated by short interfering RNA and pharmacologically. In SHR, Noxs were localized in specific subcellular regions: Nox1 in plasma membrane and Nox4 in ER. In SHR, oxidative stress was associated with increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins. Inhibition of Nox1 (NoxA1ds), Nox1/4 (GKT137831), and ER stress (4-phenylbutyric acid/tauroursodeoxycholic acid) normalized SHR vascular reactive oxygen species generation. GKT137831 reduced IRE1α sulfenylation and XBP1 (X-box binding protein 1) splicing in SHR. Increased VSMC proliferation in SHR was normalized by GKT137831, 4-phenylbutyric acid, and STF083010 (IRE1-XBP1 disruptor). Hypercontractility in the stroke-prone SHR was attenuated by 4-phenylbutyric acid. We demonstrate that protein hyperoxidation in hypertension is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4. The IRE1-XBP1 pathway of the ER stress response is regulated by Nox4/reactive oxygen species and plays a role in the hyperproliferative VSMC phenotype in SHR. Our study highlights the importance of Nox subcellular compartmentalization and interplay between cytoplasmic reactive oxygen species and ER stress response, which contribute to the VSMC oxidative proteome and vascular dysfunction in hypertension. © 2018 American Heart Association, Inc.

Increased carbonylation, protein aggregation and apoptosis in the spinal cord of mice with experimental autoimmune encephalomyelitis

PubMed Central

Dasgupta, Anushka; Zheng, Jianzheng; Perrone-Bizzozero, Nora I.; Bizzozero, Oscar A.

2013-01-01

Previous work from our laboratory implicated protein carbonylation in the pathophysiology of both MS (multiple sclerosis) and its animal model EAE (experimental autoimmune encephalomyelitis). Subsequent in vitro studies revealed that the accumulation of protein carbonyls, triggered by glutathione deficiency or proteasome inhibition, leads to protein aggregation and neuronal cell death. These findings prompted us to investigate whether their association can be also established in vivo. In the present study, we characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of MOG (myelin-oligodendrocyte glycoprotein)35–55 peptide-induced EAE in C57BL/6 mice. The results show that protein carbonyls accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. We also show a temporal correlation between protein carbonylation (but not oxidative stress) and apoptosis. Furthermore, carbonyl levels are significantly higher in apoptotic cells than in live cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are present during the course of EAE. The LC3 (microtubule-associated protein light chain 3)-II/LC3-I ratio is significantly reduced in both acute and chronic EAE indicating reduced autophagy and explaining why aggresomes accumulate in this disorder. Taken together, the results of the present study suggest a link between protein oxidation and neuronal/glial cell death in vivo, and also demonstrate impaired proteostasis in this widely used murine model of MS. PMID:23489322

Thiopurines Induce Oxidative Stress in T-Lymphocytes: A Proteomic Approach

PubMed Central

Misdaq, Misbah; Ziegler, Sonia; von Ahsen, Nicolas; Asif, Abdul R.

2015-01-01

Thiopurines are extensively used immunosuppressants for the treatment of inflammatory bowel disease (IBD). The polymorphism of thiopurine S-methyltransferase (TPMT) influences thiopurine metabolism and therapy outcome. We used a TPMT knockdown (kd) model of human Jurkat T-lymphocytes cells to study the effects of treatment with 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) on proteome and phosphoproteome. We identified thirteen proteins with altered expression and nine proteins with altered phosphorylation signals. Three proteins (THIO, TXD17, and GSTM3) with putative functions in cellular oxidative stress responses were altered by 6-TG treatment and another protein PRDX3 was differentially phosphorylated in TPMT kd cells. Furthermore, reactive oxygen species (ROS) assay results were consistent with a significant induction of oxidative stress by both TPMT knockdown and thiopurine treatments. Immunoblot analyses showed treatment altered expression of key antioxidant enzymes (i.e., SOD2 and catalase) in both wt and kd groups, while SOD1 was downregulated by 6-TG treatment and TPMT knockdown. Collectively, increased oxidative stress might be a mechanism involved in thiopurine induced cytotoxicity and adverse effects (i.e., hepatotoxicity) and an antioxidant cotherapy might help to combat this. Results highlight the significance of oxidative stress in thiopurines' actions and could have important implications for the treatment of IBD patients. PMID:25873760

Is fibromyalgia-related oxidative stress implicated in the decline of physical and mental health status?

PubMed

La Rubia, Mercedes; Rus, Alma; Molina, Francisco; Del Moral, M Luisa

2013-01-01

Fibromyalgia (FM) is a form of non-articular rheumatism characterised by chronic widespread musculoskeletal aching. Although some works have investigated the possible role of oxidative stress in the pathophysiology of FM, none has analysed a significant number of oxidative markers in the same patients. Consequently, we have performed an exhaustive study of the oxidative/antioxidative status in FM patients and healthy controls, as well as the relationship with FM clinical parameters. In 45 female patients and 25 age-matched controls, we investigated the oxidative (lipid and protein peroxidation, and oxidative DNA damage) and antioxidative status (total antioxidant capacity (TAC), and antioxidant enzyme activities and compounds). Functional capacity and musculoskeletal pain were assessed by Fibromyalgia Impact Questionnaire (FIQ) and Visual Analogue Scale (VAS), respectively. The physical (PCS-12) and mental (MCS-12) health status was evaluated by SF-12. A significant increase in oxidative DNA damage and protein carbonyl content was found in FM patients vs. controls, as well as in antioxidant compounds such as copper and ceruloplasmin. Patients had diminished levels of TAC and zinc. Enzyme activities of superoxide dismutase, glutathione peroxidase, and catalase were lower in FM patients. Significant correlations were observed in patients between oxidative DNA damage and MCS-12, and zinc and PCS-12. These findings reveal an imbalance between oxidants and antioxidants in FM patients. The lower antioxidant enzyme activities may lead to oxidative stress through the oxidation of DNA and proteins, which may affect the health status of FM patients.

Treatment with the cysteine precursor l-2-oxothiazolidine-4-carboxylate (OTC) implicates taurine deficiency in severity of dystropathology in mdx mice.

PubMed

Terrill, Jessica R; Boyatzis, Amber; Grounds, Miranda D; Arthur, Peter G

2013-09-01

Oxidative stress has been implicated in the pathology of the lethal skeletal muscle disease Duchenne muscular dystrophy (DMD), and various antioxidants have been investigated as a potential therapy. Recently, treatment of the mdx mouse model for DMD with the antioxidant and cysteine and glutathione (GSH) precursor n-acetylcysteine (NAC) was shown to decrease protein thiol oxidation and improve muscle pathology and ex vivo muscle strength. This study further investigates the mechanism for the benefits of NAC on dystrophic muscle by administering l-2-oxothiazolidine-4-carboxylate (OTC) which also upregulates intracellular cysteine and GSH, but does not directly function as an antioxidant. We observed that OTC, like NAC, decreases protein thiol oxidation, decreases pathology and increases strength, suggesting that the both NAC and OTC function via increasing cysteine and GSH content of dystrophic muscle. We demonstrate that mdx muscle is not deficient in either cysteine or GSH and that these are not increased by OTC treatment. However, we show that dystrophic muscle of 12 week old mdx mice is deficient in taurine, a by-product of disposal of excess cysteine, a deficiency that is ameliorated by OTC treatment. These data suggest that in dystrophic muscles, apart from the strong association of increased oxidative stress and protein thiol oxidation with dystropathology, another major issue is an insufficiency in taurine that can be corrected by increasing the availability of cysteine. This study provides new insight into the molecular mechanism underlying the benefits of NAC in muscular dystrophy and supports the use of OTC as an alternative drug for potential clinical applications to DMD. Copyright © 2013 Elsevier Ltd. All rights reserved.

Oxidative Stress Markers Patients with Parotid Gland Tumors: A Pilot Study

PubMed Central

Misiolek, Maciej; Pasinski, Bartlomiej; Soszynski, Miroslaw; Adamczyk-Sowa, Monika

2018-01-01

Salivary gland tumors account for 3–6% of tumors of the head and neck. About 80% of salivary gland tumors occur in parotid glands. Oxidative stress (OS) is implicated in the origin, development, and whole-body effects of various tumors. There are no data on the occurrence of OS in the parotid gland tumors. The aim of this study was to ascertain if whole-body OS accompanies parotid gland tumors, based first of all on oxidative modifications of blood serum proteins and other markers of OS in the serum of the patients. The group studied included 17 patients with pleomorphic adenoma, 9 patients with Warthin's tumor, 8 patients with acinic cell carcinoma, and 24 age-matched controls. We found increased concentration of interleukin 4 in patients with acinic cell carcinoma, decreased plasma thiols, increased AOPP concentration, and decreased FRAP of blood serum in all groups of the patients while protein oxidative modifications assessed fluorimetrically, protein carbonyls, protein nitration, malondialdehyde concentration, and serum ABTS⁎-scavenging capacity were unchanged. These data indicate the occurrence of OS in patients with parotid gland tumors and point to various sensitivities of OS markers. PMID:29651432

Proteomic pathway analysis of the hippocampus in schizophrenia and bipolar affective disorder implicates 14-3-3 signaling, aryl hydrocarbon receptor signaling, and glucose metabolism: potential roles in GABAergic interneuron pathology.

PubMed

Schubert, Klaus Oliver; Föcking, Melanie; Cotter, David R

2015-09-01

Neuropathological changes of the hippocampus have been associated with psychotic disorders such as schizophrenia and bipolar disorder. Recent work has particularly implicated hippocampal GABAergic interneurons in the pathophysiology of these diseases. However, the molecular mechanisms underlying structural and cellular hippocampal pathology remain poorly understood. We used data from comprehensive difference-in-gel electrophoresis (2-D DIGE) investigations of postmortem human hippocampus of people with schizophrenia and bipolar disorder, covering the acidic (isoelectric point (pI) between pH4 and 7) and, separately, the basic (pI between pH6 and 11) sub-proteome, for Ingenuity Pathway Analysis (IPA) of implicated protein networks and pathways. Comparing disease and control cases, we identified 58 unique differentially expressed proteins in schizophrenia, and 70 differentially expressed proteins in bipolar disorder, using mass spectrometry. IPA implicated, most prominently, 14-3-3 and aryl hydrocarbon receptor signaling in schizophrenia, and gluconeogenesis/glycolysis in bipolar disorder. Both disorders were characterized by alterations of proteins involved in the oxidative stress response, mitochondrial function, and protein-endocytosis, -trafficking, -degradation, and -ubiquitination. These findings are interpreted with a focus on GABAergic interneuron pathology in the hippocampus. Copyright © 2015 Elsevier B.V. All rights reserved.

Reactivation of oxidized PTP1B and PTEN by Thioredoxin 1

PubMed Central

Schwertassek, Ulla; Haque, Aftabul; Krishnan, Navasona; Greiner, Romy; Weingarten, Lars; Dick, Tobias P.; Tonks, Nicholas K.

2014-01-01

The transient inactivation of protein phosphatases contributes to the efficiency and temporal control of kinase-dependent signal transduction. In particular, members of the protein tyrosine phosphatase family are known to undergo reversible oxidation of their active site cysteine. The thiol oxidation step requires activation of co-localized NADPH oxidases and is mediated by locally produced ROS, in particular H2O2. How oxidized phosphatases are returned to the reduced active state is less well studied. Both major thiol reductive systems, the thioredoxin and the glutathione systems, have been implicated in the reactivation of phosphatases. Here, we show that the protein tyrosine phosphatase PTP1B and the dual-specificity phosphatase PTEN are preferentially reactivated by the thioredoxin system. We show that inducible depletion of TRX1 slows down PTEN re-activation in intact living cells. Finally, using a mechanism-based trapping approach we demonstrate direct thiol disulfide exchange between the active sites of thioredoxin and either phosphatase. The application of thioredoxin trapping mutants represents a complementary approach to direct assays of PTP oxidation in elucidating the significance of redox regulation of PTP function in the control of cell signaling. PMID:24976139

Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation

PubMed Central

Liu, Lijun; Nam, Minwoo; Fan, Wei; Akie, Thomas E.; Hoaglin, David C.; Gao, Guangping; Keaney, John F.; Cooper, Marcus P.

2014-01-01

Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation. PMID:24430182

Alterations in nonenzymatic biochemistry in uremia: origin and significance of "carbonyl stress" in long-term uremic complications.

PubMed

Miyata, T; van Ypersele de Strihou, C; Kurokawa, K; Baynes, J W

1999-02-01

Advanced glycation end products (AGEs), formed during Maillard or browning reactions by nonenzymatic glycation and oxidation (glycoxidation) of proteins, have been implicated in the pathogenesis of several diseases, including diabetes and uremia. AGEs, such as pentosidine and carboxymethyllysine, are markedly elevated in both plasma proteins and skin collagen of uremic patients, irrespective of the presence of diabetes. The increased chemical modification of proteins is not limited to AGEs, because increased levels of advanced lipoxidation end products (ALEs), such as malondialdehydelysine, are also detected in plasma proteins in uremia. The accumulation of AGEs and ALEs in uremic plasma proteins is not correlated with increased blood glucose or triglycerides, nor is it determined by a decreased removal of chemically modified proteins by glomerular filtration. It more likely results from increased plasma concentrations of small, reactive carbonyl precursors of AGEs and ALEs, such as glyoxal, methylglyoxal, 3-deoxyglucosone, dehydroascorbate, and malondialdehyde. Thus, uremia may be described as a state of carbonyl overload or "carbonyl stress" resulting from either increased oxidation of carbohydrates and lipids (oxidative stress) or inadequate detoxification or inactivation of reactive carbonyl compounds derived from both carbohydrates and lipids by oxidative and nonoxidative chemistry. Carbonyl stress in uremia may contribute to the long-term complications associated with chronic renal failure and dialysis, such as dialysis-related amyloidosis and accelerated atherosclerosis. The increased levels of AGEs and ALEs in uremic blood and tissue proteins suggest a broad derangement in the nonenzymatic biochemistry of both carbohydrates and lipids.

Abiotic Protein Fragmentation by Manganese Oxide: Implications for a Mechanism to Supply Soil Biota with Oligopeptides.

PubMed

Reardon, Patrick N; Chacon, Stephany S; Walter, Eric D; Bowden, Mark E; Washton, Nancy M; Kleber, Markus

2016-04-05

The ability of plants and microorganisms to take up organic nitrogen in the form of free amino acids and oligopeptides has received increasing attention over the last two decades, yet the mechanisms for the formation of such compounds in soil environments remain poorly understood. We used Nuclear Magnetic Resonance (NMR) and Electron Paramagnetic Resonance (EPR) spectroscopies to distinguish the reaction of a model protein with a pedogenic oxide (Birnessite, MnO2) from its response to a phyllosilicate (Kaolinite). Our data demonstrate that birnessite fragments the model protein while kaolinite does not, resulting in soluble peptides that would be available to soil biota and confirming the existence of an abiotic pathway for the formation of organic nitrogen compounds for direct uptake by plants and microorganisms. The absence of reduced Mn(II) in the solution suggests that birnessite acts as a catalyst rather than an oxidant in this reaction. NMR and EPR spectroscopies are shown to be valuable tools to observe these reactions and capture the extent of protein transformation together with the extent of mineral response.

The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages

PubMed Central

Lane, Amanda E.; Tan, Joanne T. M.; Hawkins, Clare L.; Heather, Alison K.; Davies, Michael J.

2010-01-01

MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate by hydrogen peroxide to HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid) respectively. Specificity constants indicate that SCN− is a major substrate for MPO. HOSCN is also a major oxidant generated by other peroxidases including salivary, gastric and eosinophil peroxidases. While HOCl and HOBr are powerful oxidizing agents, HOSCN is a less reactive, but more specific, oxidant which targets thiols and especially low pKa species. In the present study we show that HOSCN targets cysteine residues present in PTPs (protein tyrosine phosphatases) with this resulting in a loss of PTP activity for the isolated enzyme, in cell lysates and intact J774A.1 macrophage-like cells. Inhibition also occurs with MPO-generated HOCl and HOBr, but is more marked with MPO-generated HOSCN, particularly at longer incubation times. This inhibition is reversed by dithiothreitol, particularly at early time points, consistent with the reversible oxidation of the active site cysteine residue to give either a cysteine–SCN adduct or a sulfenic acid. Inhibition of PTP activity is associated with increased phosphorylation of p38a and ERK2 (extracellular-signal-regulated kinase 2) as detected by Western blot analysis and phosphoprotein arrays, and results in altered MAPK (mitogen-activated protein kinase) signalling. These data indicate that the highly selective targeting of some protein thiols by HOSCN can result in perturbation of cellular phosphorylation and altered cell signalling. These changes occur with (patho)physiological concentrations of SCN− ions, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage, particularly in smokers who have elevated plasma levels of SCN−. PMID:20528774

The Candida albicans stress response gene Stomatin-Like Protein 3 is implicated in ROS-induced apoptotic-like death of yeast phase cells

PubMed Central

Salcedo, Eugenia C.

2018-01-01

The ubiquitous presence of SPFH (Stomatin, Prohibitin, Flotillin, HflK/HflC) proteins in all domains of life suggests that their function would be conserved. However, SPFH functions are diverse with organism-specific attributes. SPFH proteins play critical roles in physiological processes such as mechanosensation and respiration. Here, we characterize the stomatin ORF19.7296/SLP3 in the opportunistic human pathogen Candida albicans. Consistent with the localization of stomatin proteins, a Slp3p-Yfp fusion protein formed visible puncta along the plasma membrane. We also visualized Slp3p within the vacuolar lumen. Slp3p primary sequence analyses identified four putative S-palmitoylation sites, which may facilitate membrane localization and are conserved features of stomatins. Plasma membrane insertion sequences are present in mammalian and nematode SPFH proteins, but are absent in Slp3p. Strikingly, Slp3p was present in yeast cells, but was absent in hyphal cells, thus categorizing it as a yeast-phase specific protein. Slp3p membrane fluorescence significantly increased in response to cellular stress caused by plasma membrane, cell wall, oxidative, or osmotic perturbants, implicating SLP3 as a general stress-response gene. A slp3Δ/Δ homozygous null mutant had no detected phenotype when slp3Δ/Δ mutants were grown in the presence of a variety of stress agents. Also, we did not observe a defect in ion accumulation, filamentation, endocytosis, vacuolar structure and function, cell wall structure, or cytoskeletal structure. However, SLP3 over-expression triggered apoptotic-like death following prolonged exposure to oxidative stress or when cells were induced to form hyphae. Our findings reveal the cellular localization of Slp3p, and for the first time associate Slp3p function with the oxidative stress response. PMID:29389961

Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide sensitive iron-sulphur cluster

PubMed Central

Smith, Laura J.; Stapleton, Melanie R.; Fullstone, Gavin J. M.; Crack, Jason C.; Thomson, Andrew J.; Le Brun, Nick E.; Hunt, Debbie M.; Harvey, Evelyn; Adinolfi, Salvatore; Buxton, Roger S.; Green, Jeffrey

2010-01-01

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. Here it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however in the presence of apo-WhiB1 transcription was severely inhibited, irrespective of the presence or absence of the CRP protein Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections. PMID:20929442

Factors Affecting Pro- and Anti-Oxidant Properties of Fragments of the b-Protein Precursor (bPP): Implication for Alzheimer's Disease.

PubMed

Andorn, Anne C.; Kalaria, Rajesh N.

2000-06-01

Oxidative stress may have a key pathogenetic role in neurodegenerative diseases including Alzheimer's disease (AD). While there is evidence that some amyloid-b (Ab) peptides can initiate oxidative stress at micromolar doses, there is also some evidence that oxidative stress increases the concentration of the b-protein precursor (bPP) and the potential for increased formation of the Ab peptides. The following studies were performed to test the hypothesis that fragments of bPP could be antioxidants and hence that oxidative stress might be an early event in AD. We found that several fragments of bPP, including the Ab peptides, inhibit ascorbate-stimulated lipid peroxidation (ASLP) in membrane fragment preparations of postmortem human brain. In contrast, other fragments of bPP enhance ASLP. These data indicate that bPP or fragments of bPP could play a key role in the redox status of cells and that alterations in bPP processing could have profound effects on the cellular response to oxidative stress.

Redox Regulation in Amyotrophic Lateral Sclerosis

PubMed Central

Parakh, Sonam; Spencer, Damian M.; Halloran, Mark A.; Soo, Kai Y.; Atkin, Julie D.

2013-01-01

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results from the death of upper and lower motor neurons. Due to a lack of effective treatment, it is imperative to understand the underlying mechanisms and processes involved in disease progression. Regulations in cellular reduction/oxidation (redox) processes are being increasingly implicated in disease. Here we discuss the possible involvement of redox dysregulation in the pathophysiology of ALS, either as a cause of cellular abnormalities or a consequence. We focus on its possible role in oxidative stress, protein misfolding, glutamate excitotoxicity, lipid peroxidation and cholesterol esterification, mitochondrial dysfunction, impaired axonal transport and neurofilament aggregation, autophagic stress, and endoplasmic reticulum (ER) stress. We also speculate that an ER chaperone protein disulphide isomerase (PDI) could play a key role in this dysregulation. PDI is essential for normal protein folding by oxidation and reduction of disulphide bonds, and hence any disruption to this process may have consequences for motor neurons. Addressing the mechanism underlying redox regulation and dysregulation may therefore help to unravel the molecular mechanism involved in ALS. PMID:23533690

The impact of the unfolded protein response on human disease

PubMed Central

Wang, Shiyu

2012-01-01

A central function of the endoplasmic reticulum (ER) is to coordinate protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease. PMID:22733998

Post-translational Regulation of Neuronal Nitric Oxide Synthase: Implications for sympatho-excitatory states

PubMed Central

Sharma, Neeru M; Patel, Kaushik P

2017-01-01

Introduction Nitric oxide (NO) synthesized via neuronal nitric oxide synthase (nNOS) plays a significant role in regulation/modulation of autonomic control of circulation. Various pathological states are associated with diminished nNOS expression and blunted autonomic effects of NO in the central nervous system (CNS) including heart failure, hypertension, diabetes mellitus, chronic renal failure etc. Therefore, elucidation of the molecular mechanism/s involved in dysregulation of nNOS is essential to understand the pathogenesis of increased sympathoexcitation in these diseased states. Areas Covered nNOS is a highly regulated enzyme, being regulated at transcriptional and posttranslational levels via protein-protein interactions and modifications viz. phosphorylation, ubiquitination, and sumoylation. The enzyme activity of nNOS also depends on the optimal concentration of substrate, cofactors and association with regulatory proteins. This review focuses on the posttranslational regulation of nNOS in the context of normal and diseased states within the CNS. Expert Opinion Gaining insight into the mechanism/s involved in the regulation of nNOS would provide novel strategies for manipulating nNOS directed therapeutic modalities in the future, including catalytically active dimer stabilization and protein-protein interactions with intracellular protein effectors. Ultimately, this is expected to provide tools to improve autonomic dysregulation in various diseases such as heart failure, hypertension, and diabetes. PMID:27885874

Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

PubMed Central

Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

2014-01-01

Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters.

PubMed

Du, Wei; Rani, Reena; Sipple, Jared; Schick, Jonathan; Myers, Kasiani C; Mehta, Parinda; Andreassen, Paul R; Davies, Stella M; Pang, Qishen

2012-05-03

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Oxidative stress in Alzheimer disease and mild cognitive impairment: evidence from human data provided by redox proteomics.

PubMed

Swomley, Aaron M; Butterfield, D Allan

2015-10-01

Alzheimer disease (AD) is a neurodegenerative disease with many known pathological features, yet there is still much debate into the exact cause and mechanisms for progression of this degenerative disorder. The amyloid-beta (Aβ)-induced oxidative stress hypothesis postulates that it is the oligomeric Aβ that inserts into membrane systems to initiate much of the oxidative stress observed in brain during the progression of the disease. In order to study the effects of oxidative stress on tissue from patients with AD and amnestic mild cognitive impairment (MCI), we have developed a method called redox proteomics that identifies specific brain proteins found to be selectively oxidized. Here, we discuss experimental findings of oxidatively modified proteins involved in three key cellular processes implicated in the pathogenesis of AD progression: energy metabolism, cell signaling and neurotransmission, as well as the proteasomal degradation pathways and antioxidant response systems. These proteomics studies conducted by our laboratory and others in the field shed light on the molecular changes imposed on the cells of AD and MCI brain, through the deregulated increase in oxidative/nitrosative stress inflicted by Aβ and mitochondrial dysfunction.

Dietary glutamate reduces systemic but not intestinal leucine oxidation in protein malnourished piglets

USDA-ARS?s Scientific Manuscript database

The methionine (Met) metabolic cycle is critical for normal cell functions. Met cycle disruption has been implicated in disease, such as alcoholic liver disease (ALD) and multiple sclerosis (MS). Studies in animal models of ALD and MS have shown that the Met metabolite methylthioadenosine (MTA) has ...

Nutrients and Oxidative Stress: Friend or Foe?

PubMed Central

Tan, Bee Ling; Liew, Winnie-Pui-Pui

2018-01-01

There are different types of nutritionally mediated oxidative stress sources that trigger inflammation. Much information indicates that high intakes of macronutrients can promote oxidative stress and subsequently contribute to inflammation via nuclear factor-kappa B- (NF-κB-) mediated cell signaling pathways. Dietary carbohydrates, animal-based proteins, and fats are important to highlight here because they may contribute to the long-term consequences of nutritionally mediated inflammation. Oxidative stress is a central player of metabolic ailments associated with high-carbohydrate and animal-based protein diets and excessive fat consumption. Obesity has become an epidemic and represents the major risk factor for several chronic diseases, including diabetes, cardiovascular disease (CVD), and cancer. However, the molecular mechanisms of nutritionally mediated oxidative stress are complex and poorly understood. Therefore, this review aimed to explore how dietary choices exacerbate or dampen the oxidative stress and inflammation. We also discussed the implications of oxidative stress in the adipocyte and glucose metabolism and obesity-associated noncommunicable diseases (NCDs). Taken together, a better understanding of the role of oxidative stress in obesity and the development of obesity-related NCDs would provide a useful approach. This is because oxidative stress can be mediated by both extrinsic and intrinsic factors, hence providing a plausible means for the prevention of metabolic disorders. PMID:29643982

Nutrients and Oxidative Stress: Friend or Foe?

PubMed

Tan, Bee Ling; Norhaizan, Mohd Esa; Liew, Winnie-Pui-Pui

2018-01-01

There are different types of nutritionally mediated oxidative stress sources that trigger inflammation. Much information indicates that high intakes of macronutrients can promote oxidative stress and subsequently contribute to inflammation via nuclear factor-kappa B- (NF- κ B-) mediated cell signaling pathways. Dietary carbohydrates, animal-based proteins, and fats are important to highlight here because they may contribute to the long-term consequences of nutritionally mediated inflammation. Oxidative stress is a central player of metabolic ailments associated with high-carbohydrate and animal-based protein diets and excessive fat consumption. Obesity has become an epidemic and represents the major risk factor for several chronic diseases, including diabetes, cardiovascular disease (CVD), and cancer. However, the molecular mechanisms of nutritionally mediated oxidative stress are complex and poorly understood. Therefore, this review aimed to explore how dietary choices exacerbate or dampen the oxidative stress and inflammation. We also discussed the implications of oxidative stress in the adipocyte and glucose metabolism and obesity-associated noncommunicable diseases (NCDs). Taken together, a better understanding of the role of oxidative stress in obesity and the development of obesity-related NCDs would provide a useful approach. This is because oxidative stress can be mediated by both extrinsic and intrinsic factors, hence providing a plausible means for the prevention of metabolic disorders.

Protein oxidation and aging. I. Difficulties in measuring reactive protein carbonyls in tissues using 2,4-dinitrophenylhydrazine.

PubMed

Cao, G; Cutler, R G

1995-06-20

A current hypothesis explaining the aging process implicates the accumulation of oxidized protein in animal tissues. This hypothesis is based on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent loss of enzyme function. This hypothesis is also supported by the report of a novel effect of N-tert-butyl-alpha-phenylnitrone (PBN) in reversing these age-dependent changes. Here we specifically study the method that was used to measure reactive protein carbonyls in tissues. This method uses 2,4-dinitrophenylhydrazine (DNPH) and includes a washing procedure. Our results indicate that reactive protein carbonyls in normal crude tissue extracts cannot be reliably measured by this method, although it does reliably measure reactive carbonyls in purified proteins which have been oxidatively modified in vitro. The nucleic acids in tissues could be a major problem encountered in the assay. Using the streptomycin sulfate treatment combined with a dialysis step, we were successful in removing most nucleic acids from a crude tissue extract, but then the reactive carbonyl level in the crude tissue extract was too low to be reliably measured. This streptomycin sulfate treatment procedure, however, had no effect on the reactive carbonyl measurement of an oxidized protein sample. The unwashed free DNPH was another major problem in the assay because of its very strong absorption around 370 nm, where reactive carbonyls were quantitated. Nevertheless, on using the procedure described in the literature to measure total "reactive carbonyls" in rat liver and gerbil brain cortex, no change with age or PBN treatment was found. Then, we investigated a HPLC procedure which uses sodium dodecyl sulfate in the mobile phase but this was also found to be unsuitable for the reactive protein carbonyl assay in tissues.

ATM directs DNA damage responses and proteostasis via genetically separable pathways.

PubMed

Lee, Ji-Hoon; Mand, Michael R; Kao, Chung-Hsuan; Zhou, Yi; Ryu, Seung W; Richards, Alicia L; Coon, Joshua J; Paull, Tanya T

2018-01-09

The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATM causes ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. We genetically separated the activation of ATM by DNA damage from that by oxidative stress using separation-of-function mutations. We found that deficient activation of ATM by the Mre11-Rad50-Nbs1 complex and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATM had minimal effects on DNA damage-related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of a variant ATM incapable of activation by oxidative stress resulted in widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATM activation and its effects on cellular metabolism and DNA damage responses in human cells and implicate ATM in the control of protein homeostasis. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Chemical hybridizing agent SQ-1-induced male sterility in Triticum aestivum L.: a comparative analysis of the anther proteome.

PubMed

Liu, Hongzhan; Zhang, Gaisheng; Wang, Junsheng; Li, Jingjing; Song, Yulong; Qiao, Lin; Niu, Na; Wang, Junwei; Ma, Shoucai; Li, Lili

2018-01-05

Heterosis is widely used to increase the yield of many crops. However, as wheat is a self-pollinating crop, hybrid breeding is not so successful in this organism. Even though male sterility induced by chemical hybridizing agents is an important aspect of crossbreeding, the mechanisms by which these agents induce male sterility in wheat is not well understood. We performed proteomic analyses using the wheat Triticum aestivum L.to identify those proteins involved in physiological male sterility (PHYMS) induced by the chemical hybridizing agent CHA SQ-1. A total of 103 differentially expressed proteins were found by 2D-PAGE and subsequently identified by MALDI-TOF/TOF MS/MS. In general, these proteins had obvious functional tendencies implicated in carbohydrate metabolism, oxidative stress and resistance, protein metabolism, photosynthesis, and cytoskeleton and cell structure. In combination with phenotypic, tissue section, and bioinformatics analyses, the identified differentially expressed proteins revealed a complex network behind the regulation of PHYMS and pollen development. Accordingly, we constructed a protein network of male sterility in wheat, drawing relationships between the 103 differentially expressed proteins and their annotated biological pathways. To further validate our proposed protein network, we determined relevant physiological values and performed real-time PCR assays. Our proteomics based approach has enabled us to identify certain tendencies in PHYMS anthers. Anomalies in carbohydrate metabolism and oxidative stress, together with premature tapetum degradation, may be the cause behind carbohydrate starvation and male sterility in CHA SQ-1 treated plants. Here, we provide important insight into the mechanisms underlying CHA SQ-1-induced male sterility. Our findings have practical implications for the application of hybrid breeding in wheat.

Antioxidant Effectiveness of Vegetable Powders on the Lipid and Protein Oxidative Stability of Cooked Turkey Meat Patties: Implications for Health

PubMed Central

Duthie, Garry; Campbell, Fiona; Bestwick, Charles; Stephen, Sylvia; Russell, Wendy

2013-01-01

Lipid and protein oxidation decreases the shelf-life of foods and may result in formation of end-products potentially detrimental for health. Consumer pressure to decrease the use of synthetic phenolic antioxidants has encouraged identification of alternative compounds or extracts from natural sources. We have assessed whether inclusion of dried vegetable powders improves the oxidative stability of turkey meat patties. Such powders are not only potentially-rich sources of phenolic antioxidants, but also may impart additional health benefits, as inadequate vegetable consumption is a risk factor for heart disease and several cancers. In an accelerated oxidation system, six of eleven vegetable powders significantly (p < 0.05) improved oxidative stability of patties by 20%–30% (spinach < yellow pea < onion < red pepper < green pea < tomato). Improved lipid oxidative stability was strongly correlated with the decreased formation of protein carbonyls (r = 0.747, p < 0.01). However, improved lipid stability could not be ascribed to phenolic acids nor recognized antioxidants, such as α- and γ-tocopherol, despite their significant (p < 0.01) contribution to the total antioxidant capacity of the patties. Use of chemically complex vegetable powders offers an alternative to individual antioxidants for increasing shelf-life of animal-based food products and may also provide additional health benefits associated with increased vegetable intake. PMID:23595133

Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function During Nanoparticle-Induced Oxidative Stress

DOE PAGES

Duan, Jicheng; Kodali, Vamsi K.; Gaffrey, Matthew J.; ...

2015-12-23

Engineered nanoparticles (ENPs) are emerging functional materials increasingly utilized for commercial and medical applications. Due to the potential hazard effects of ENPs to human health, it is significant to assess and understand the underlying mechanisms of nanotoxicity. Here, we investigate protein S-glutathionylation (SSG) as an underlying regulatory mechanism for ENP-induced oxidative stress in macrophages by applying a recently developed quantitative redox proteomics approach for site-specific measurements of SSG. Three high-volume production ENPs (SiO 2, Fe 3O 4 and CoO) were selected as representative ENPs with low, moderate, and high reactive oxygen species (ROS) activity, respectively. Among these nanoparticles, we observemore » that CoO led to the most significant dose-dependent oxidative stress and increase of protein SSG modifications in macrophages. Our site-specific SSG changes highlighted a broad set of redox sensitive proteins and their specific Cys residues potentially implicated in stress response. Functional analysis revealed that the most significantly enriched functional categories for SSG-modified proteins were stress response, cellular structure change, and cell death or survival. Moreover, ENPs-induce oxidative stress levels (CoO > Fe 3O 4 > SiO 2) were found to correlate well with the levels of impairment of macrophage phagocytic activity and the overall degrees of increases in SSG. RNA silencing knockdown experiment of glutaredoxin 1 (Grx1) also led to a decreased phagocytic activity in macrophages, which suggested a regulatory role of SSG in phagocytosis. Together, the results provided valuable insights of protein SSG as a potential regulatory mechanism in response to nanomaterial-induced oxidative stress and immunity dysfunction.« less

Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function During Nanoparticle-Induced Oxidative Stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duan, Jicheng; Kodali, Vamsi K.; Gaffrey, Matthew J.

Engineered nanoparticles (ENPs) are emerging functional materials increasingly utilized for commercial and medical applications. Due to the potential hazard effects of ENPs to human health, it is significant to assess and understand the underlying mechanisms of nanotoxicity. Here, we investigate protein S-glutathionylation (SSG) as an underlying regulatory mechanism for ENP-induced oxidative stress in macrophages by applying a recently developed quantitative redox proteomics approach for site-specific measurements of SSG. Three high-volume production ENPs (SiO 2, Fe 3O 4 and CoO) were selected as representative ENPs with low, moderate, and high reactive oxygen species (ROS) activity, respectively. Among these nanoparticles, we observemore » that CoO led to the most significant dose-dependent oxidative stress and increase of protein SSG modifications in macrophages. Our site-specific SSG changes highlighted a broad set of redox sensitive proteins and their specific Cys residues potentially implicated in stress response. Functional analysis revealed that the most significantly enriched functional categories for SSG-modified proteins were stress response, cellular structure change, and cell death or survival. Moreover, ENPs-induce oxidative stress levels (CoO > Fe 3O 4 > SiO 2) were found to correlate well with the levels of impairment of macrophage phagocytic activity and the overall degrees of increases in SSG. RNA silencing knockdown experiment of glutaredoxin 1 (Grx1) also led to a decreased phagocytic activity in macrophages, which suggested a regulatory role of SSG in phagocytosis. Together, the results provided valuable insights of protein SSG as a potential regulatory mechanism in response to nanomaterial-induced oxidative stress and immunity dysfunction.« less

Redox Control of the Human Iron-Sulfur Repair Protein MitoNEET Activity via Its Iron-Sulfur Cluster*

PubMed Central

Golinelli-Cohen, Marie-Pierre; Lescop, Ewen; Mons, Cécile; Gonçalves, Sergio; Clémancey, Martin; Santolini, Jérôme; Guittet, Eric; Blondin, Geneviève; Latour, Jean-Marc; Bouton, Cécile

2016-01-01

Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, mNT has been implicated in cytosolic Fe-S repair of a key regulator of cellular iron homeostasis. Here, we aimed to decipher the mechanism by which mNT triggers its Fe-S repair capacity. By using tightly controlled reactions combined with complementary spectroscopic approaches, we have determined the differential roles played by both the redox state of the mNT cluster and dioxygen in cluster transfer and protein stability. We unambiguously demonstrated that only the oxidized state of the mNT cluster triggers cluster transfer to a generic acceptor protein and that dioxygen is neither required for the cluster transfer reaction nor does it affect the transfer rate. In the absence of apo-acceptors, a large fraction of the oxidized holo-mNT form is converted back to reduced holo-mNT under low oxygen tension. Reduced holo-mNT, which holds a [2Fe-2S]+ with a global protein fold similar to that of the oxidized form is, by contrast, resistant in losing its cluster or in transferring it. Our findings thus demonstrate that mNT uses an iron-based redox switch mechanism to regulate the transfer of its cluster. The oxidized state is the “active state,” which reacts promptly to initiate Fe-S transfer independently of dioxygen, whereas the reduced state is a “dormant form.” Finally, we propose that the redox-sensing function of mNT is a key component of the cellular adaptive response to help stress-sensitive Fe-S proteins recover from oxidative injury. PMID:26887944

The effects of chromium(VI) on the thioredoxin system: Implications for redox regulation

PubMed Central

Myers, Charles R.

2014-01-01

Hexavalent chromium [Cr(VI)] compounds are highly redox active and have long been recognized as potent cytotoxins and carcinogens. The intracellular reduction of Cr(VI) generates reactive Cr intermediates, which are themselves strong oxidants, as well as superoxide, hydrogen peroxide, and hydroxyl radical. These probably contribute to the oxidative damage and effects on redox-sensitive transcription factors that have been reported. However, the identification of events that initiate these signaling changes has been elusive. More recent studies show that Cr(VI) causes irreversible inhibition of thioredoxin reductase (TrxR) and oxidation of thioredoxin (Trx) and peroxiredoxin (Prx). Mitochondrial Trx2/Prx3 are more sensitive to Cr(VI) treatment than cytosolic Trx1/Prx1, although both compartments show thiol oxidation with higher doses or longer treatments. Thiol redox proteomics demonstrate that Trx2, Prx3, and Trx1 are among the most sensitive proteins in cells to Cr(VI) treatment. Their oxidation could therefore represent initiating events that have widespread implications for protein thiol redox control and for multiple aspects of redox signaling. This review summarizes the effects of Cr(VI) on the TrxR/Trx system and how these events could influence a number of downstream redox signaling systems that are influenced by Cr(VI) exposure. Some of the signaling events discussed include the activation of apoptosis signal regulating kinase and MAP kinases (p38 and JNK) and the modulation of a number of redox-sensitive transcription factors including AP-1, NF-κB, p53, and Nrf2. PMID:22542445

Macrophage Migration Inhibitory Factor is subjected to glucose modification and oxidation in Alzheimer’s Disease

PubMed Central

Kassaar, Omar; Pereira Morais, Marta; Xu, Suying; Adam, Emily L.; Chamberlain, Rosemary C.; Jenkins, Bryony; James, Tony; Francis, Paul T.; Ward, Stephen; Williams, Robert J.; van den Elsen, Jean

2017-01-01

Glucose and glucose metabolites are able to adversely modify proteins through a non-enzymatic reaction called glycation, which is associated with the pathology of Alzheimer’s Disease (AD) and is a characteristic of the hyperglycaemia induced by diabetes. However, the precise protein glycation profile that characterises AD is poorly defined and the molecular link between hyperglycaemia and AD is unknown. In this study, we define an early glycation profile of human brain using fluorescent phenylboronate gel electrophoresis and identify early glycation and oxidation of macrophage migration inhibitory factor (MIF) in AD brain. This modification inhibits MIF enzyme activity and ability to stimulate glial cells. MIF is involved in immune response and insulin regulation, hyperglycaemia, oxidative stress and glycation are all implicated in AD. Our study indicates that glucose modified and oxidised MIF could be a molecular link between hyperglycaemia and the dysregulation of the innate immune system in AD. PMID:28230058

The adaptive evolution of the mammalian mitochondrial genome

PubMed Central

da Fonseca, Rute R; Johnson, Warren E; O'Brien, Stephen J; Ramos, Maria João; Antunes, Agostinho

2008-01-01

Background The mitochondria produce up to 95% of a eukaryotic cell's energy through oxidative phosphorylation. The proteins involved in this vital process are under high functional constraints. However, metabolic requirements vary across species, potentially modifying selective pressures. We evaluate the adaptive evolution of 12 protein-coding mitochondrial genes in 41 placental mammalian species by assessing amino acid sequence variation and exploring the functional implications of observed variation in secondary and tertiary protein structures. Results Wide variation in the properties of amino acids were observed at functionally important regions of cytochrome b in species with more-specialized metabolic requirements (such as adaptation to low energy diet or large body size, such as in elephant, dugong, sloth, and pangolin, and adaptation to unusual oxygen requirements, for example diving in cetaceans, flying in bats, and living at high altitudes in alpacas). Signatures of adaptive variation in the NADH dehydrogenase complex were restricted to the loop regions of the transmembrane units which likely function as protons pumps. Evidence of adaptive variation in the cytochrome c oxidase complex was observed mostly at the interface between the mitochondrial and nuclear-encoded subunits, perhaps evidence of co-evolution. The ATP8 subunit, which has an important role in the assembly of F0, exhibited the highest signal of adaptive variation. ATP6, which has an essential role in rotor performance, showed a high adaptive variation in predicted loop areas. Conclusion Our study provides insight into the adaptive evolution of the mtDNA genome in mammals and its implications for the molecular mechanism of oxidative phosphorylation. We present a framework for future experimental characterization of the impact of specific mutations in the function, physiology, and interactions of the mtDNA encoded proteins involved in oxidative phosphorylation. PMID:18318906

Study of telomere length and different markers of oxidative stress in patients with Parkinson's disease.

PubMed

Watfa, G; Dragonas, C; Brosche, T; Dittrich, R; Sieber, C C; Alecu, C; Benetos, A; Nzietchueng, R

2011-04-01

Many studies have shown that short telomere length (TL) is associated with high oxidative stress and various age-related diseases. Parkinson's disease (PD) is an age-related disease, and although its pathogenic mechanism is uncertain, oxidative stress is believed to be implicated in this pathology. The aim of this case-control study was to assess both TL and the different markers of oxidative stress in elderly patients with PD compared to age control subjects. 20 PD patients and 15 age-matched controls, >65 years were studied. TL was measured by Southern blotting from DNA samples extracted from white blood cells. Superoxide dismutase (SOD) activity and plasma levels of total glutathione and protein carbonyls were determined. There was a trend for lower TL in PD patients: 6.06 ± 0.81 kb in PD versus 6.45 ± 0.73 kb in controls (p = 0.08). No significant difference was found between the two groups in terms of oxidative stress markers. In controls, age was the main determinant of telomere shortening (r = -0.547; p = 0.03) whereas, in PD patients, telomere shortening was mainly dependent on plasmatic concentrations of carbonyl proteins (r= -0.544; p=0.044). In PD patients, a negative association was observed between plasma carbonyl protein levels and SOD activity (r= -0.622, p=0.004). In PD, TL is shorter in presence of high oxidative stress as measured by carbonyl protein levels. The absence of telomere attrition with age among patients with PD could reflect a telomere regulation by mechanisms other than age.

JNK Activation of BIM Promotes Hepatic Oxidative Stress, Steatosis, and Insulin Resistance in Obesity.

PubMed

Litwak, Sara A; Pang, Lokman; Galic, Sandra; Igoillo-Esteve, Mariana; Stanley, William J; Turatsinze, Jean-Valery; Loh, Kim; Thomas, Helen E; Sharma, Arpeeta; Trepo, Eric; Moreno, Christophe; Gough, Daniel J; Eizirik, Decio L; de Haan, Judy B; Gurzov, Esteban N

2017-12-01

The members of the BCL-2 family are crucial regulators of the mitochondrial pathway of apoptosis in normal physiology and disease. Besides their role in cell death, BCL-2 proteins have been implicated in the regulation of mitochondrial oxidative phosphorylation and cellular metabolism. It remains unclear, however, whether these proteins have a physiological role in glucose homeostasis and metabolism in vivo. In this study, we report that fat accumulation in the liver increases c-Jun N-terminal kinase-dependent BCL-2 interacting mediator of cell death (BIM) expression in hepatocytes. To determine the consequences of hepatic BIM deficiency in diet-induced obesity, we generated liver-specific BIM-knockout (BLKO) mice. BLKO mice had lower hepatic lipid content, increased insulin signaling, and improved global glucose metabolism. Consistent with these findings, lipogenic and lipid uptake genes were downregulated and lipid oxidation enhanced in obese BLKO mice. Mechanistically, BIM deficiency improved mitochondrial function and decreased oxidative stress and oxidation of protein tyrosine phosphatases, and ameliorated activation of peroxisome proliferator-activated receptor γ/sterol regulatory element-binding protein 1/CD36 in hepatocytes from high fat-fed mice. Importantly, short-term knockdown of BIM rescued obese mice from insulin resistance, evidenced by reduced fat accumulation and improved insulin sensitivity. Our data indicate that BIM is an important regulator of liver dysfunction in obesity and a novel therapeutic target for restoring hepatocyte function. © 2017 by the American Diabetes Association.

Effects of Single and Combined Losartan and Tempol Treatments on Oxidative Stress, Kidney Structure and Function in Spontaneously Hypertensive Rats with Early Course of Proteinuric Nephropathy.

PubMed

Karanovic, Danijela; Grujic-Milanovic, Jelica; Miloradovic, Zoran; Ivanov, Milan; Jovovic, Djurdjica; Vajic, Una-Jovana; Zivotic, Maja; Markovic-Lipkovski, Jasmina; Mihailovic-Stanojevic, Nevena

2016-01-01

Oxidative stress has been widely implicated in both hypertension and chronic kidney disease (CKD). Hypertension is a major risk factor for CKD progression. In the present study we have investigated the effects of chronic single tempol (membrane-permeable radical scavenger) or losartan (angiotensin II type 1 receptor blocker) treatment, and their combination on systemic oxidative status (plasma thiobarbituric acid-reactive substances (pTBARS) production, plasma antioxidant capacity (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, pABTS), erythrocyte antioxidant enzymes activities) and kidney oxidative stress (kTBARS, kABTS, kidney antioxidant enzymes activities), kidney function and structure in spontaneously hypertensive rats (SHR) with the early course of adriamycin-induced nephropathy. Adult SHR were divided into five groups. The control group received vehicle, while the other groups received adriamycin (2 mg/kg, i.v.) twice in a 21-day interval, followed by vehicle, losartan (L,10 mg/kg/day), tempol (T,100 mg/kg/day) or combined T+L treatment (by gavage) during a six-week period. Adriamycin significantly increased proteinuria, plasma lipid peroxidation, kidney protein oxidation, nitrite excretion, matrix metalloproteinase-1 (MMP-1) protein expression and nestin immunostaining in the kidney. Also, it decreased kidney antioxidant defense, kidney NADPH oxidase 4 (kNox4) protein expression and abolished anti-inflammatory response due to significant reduction of kidney NADPH oxidase 2 (kNox2) protein expression in SHR. All treatments reduced protein-to-creatinine ratio (marker of proteinuria), pTBARS production, kidney protein carbonylation, nitrite excretion, increased antioxidant capacity and restored kidney nestin expression similar to control. Both single treatments significantly improved systemic and kidney antioxidant defense, bioavailability of renal nitric oxide, reduced kMMP-1 protein expression and renal injury, thus retarded CKD progression. Losartan improved blood pressure, as well as tubular injury and restored anti-inflammatory defense by reverting kNox2 expression to the control level. Interestingly, tempol was more successful in reducing systemic oxidative stress, proteinuria, kMMP-1 and glomerulosclerosis. However, combined treatment failed to overcome the beneficial effects of single treatments in slowing down the progression of ADR-induced nephropathy in SHR.

Inhibition of cholesterol oxidation products (COPs) formation in emulsified porcine patties by phenolic-rich avocado (Persea americana Mill.) extracts.

PubMed

Rodríguez-Carpena, Javier-Germán; Morcuende, David; Petrón, María Jesus; Estévez, Mario

2012-03-07

The effect of phenolic-rich extracts from avocado peel on the formation of cholesterol oxidation products (COPs) in porcine patties subjected to cooking and chill storage was studied. Eight COPs (7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 20α-hydroxycholesterol, 25-hydroxycholesterol, cholestanetriol, 5,6β-epoxycholesterol, and 5,6α-epoxycholesterol) were identified and quantified by GC-MS. The addition of avocado extracts (∼600 GAE/kg patty) to patties significantly inhibited the formation of COPs during cooking. Cooked control (C) patties contained a larger variety and greater amounts of COPs than the avocado-treated (T) counterparts. COPs sharply increased in cooked patties during the subsequent chilled storage. This increase was significantly higher in C patties than in the T patties. Interestingly, the amount of COPs in cooked and chilled T patties was similar to those found in cooked C patties. The mechanisms implicated in cholesterol oxidation in a processed meat product, the protective effect of avocado phenolics, and the potential implication of lipid and protein oxidation are thoroughly described in the present paper.

High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis.

PubMed

Nagaraj, R H; Sell, D R; Prabhakaram, M; Ortwerth, B J; Monnier, V M

1991-11-15

Pentosidine is a recently discovered protein crosslink, involving lysine and arginine residues linked together in an imidazo [4,5,6] pyridinium ring formed by a 5-carbon sugar during nonenzymatic browning (Maillard reaction). The presence of high ascorbate levels in the human lens and its ability to undergo nonenzymatic browning led us to investigate pentosidine formation in the aging human lens. Incubation of lens crystallins with ascorbate and its oxidation products dehydroascorbate and 2,3-diketogulonate leads progressively to the formation of pentosidine crosslinks in the presence of oxygen. Under nitrogen, however, pentosidine forms only from 2,3-diketogulonate or xylosone, a degradation product of 2,3-diketogulonate. A high correlation between pentosidine crosslinks and the degree of lens pigmentation is noted in cataractous lenses. Pentosidine is found to be primarily associated with alpha-crystallin fractions of 300-5000 kDa. These results suggest that redox imbalance in cellular senescent systems such as the ocular lens may lead to irreversible ascorbate oxidation and protein crosslinking by xylosone. This mechanism may play an important role in the pathogenesis of "brunescent" cataracts.

Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome-wide turnover

PubMed Central

Swaney, Danielle L; Rodríguez-Mias, Ricard A; Villén, Judit

2015-01-01

Ubiquitylation is an essential post-translational modification that regulates numerous cellular processes, most notably protein degradation. Ubiquitin can itself be phosphorylated at nearly every serine, threonine, and tyrosine residue. However, the effect of this modification on ubiquitin function is largely unknown. Here, we characterized the effects of phosphorylation of yeast ubiquitin at serine 65 in vivo and in vitro. We find this post-translational modification to be regulated under oxidative stress, occurring concomitantly with the restructuring of the ubiquitin landscape into a highly polymeric state. Phosphomimetic mutation of S65 recapitulates the oxidative stress phenotype, causing a dramatic accumulation of ubiquitylated proteins and a proteome-wide reduction of protein turnover rates. Importantly, this mutation impacts ubiquitin chain disassembly, chain linkage distribution, ubiquitin interactions, and substrate targeting. These results demonstrate that phosphorylation is an additional mode of ubiquitin regulation with broad implications in cellular physiology. PMID:26142280

Crystallization and preliminary crystallographic studies of Hyp-1, a St John’s wort protein implicated in the biosynthesis of hypericin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fernandes, Humberto; Konieczna, Malgorzata; Kolodziejczyk, Robert

2008-05-01

The Hyp-1 protein suggested to be a phenolic oxidative-coupling enzyme involved in the biosynthesis of hypericin, a medicinally important natural compound found in St John’s wort (H. perforatum), has been expressed, purified and prepared in single-crystal form. According to a debated hypothesis, the biosynthesis from emodin of the medicinally important natural compound hypericin is catalyzed in St John’s wort (Hypericum perforatum) by the phenolic oxidative-coupling protein Hyp-1. Recombinant St John’s wort Hyp-1 has been overexpressed in Escherichia coli and obtained in single-crystal form. The crystals belong to the orthorhombic system, space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters amore » = 37.5, b = 76.7, c = 119.8 Å, contain two protein molecules in the asymmetric unit and diffract X-rays to 1.73 Å resolution.« less

Attachment of Pathogenic Prion Protein to Model Oxide Surfaces

PubMed Central

Jacobson, Kurt H.; Kuech, Thomas R.; Pedersen, Joel A.

2014-01-01

Prions are the infectious agents in the class of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies, which affect humans, deer, sheep, and cattle. Prion diseases of deer and sheep can be transmitted via environmental routes, and soil is has been implicated in the transmission of these diseases. Interaction with soil particles is expected to govern the transport, bioavailability and persistence of prions in soil environments. A mechanistic understanding of prion interaction with soil components is critical for understanding the behavior of these proteins in the environment. Here, we report results of a study to investigate the interactions of prions with model oxide surfaces (Al2O3, SiO2) using quartz crystal microbalance with dissipation monitoring and optical waveguide light mode spectroscopy. The efficiency of prion attachment to Al2O3 and SiO2 depended strongly on pH and ionic strength in a manner consistent with electrostatic forces dominating interaction with these oxides. The N-terminal portion of the protein appeared to facilitate attachment to Al2O3 under globally electrostatically repulsive conditions. We evaluated the utility of recombinant prion protein as a surrogate for prions in attachment experiments and found that its behavior differed markedly from that of the infectious agent. Our findings suggest that prions preferentially associate with positively charged mineral surfaces in soils (e.g., iron and aluminum oxides). PMID:23611152

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR*

PubMed Central

Kafle, Prapti; Amoh, Amanda N.; Reaves, Jocelyn M.; Suneby, Emma G.; Tutunjian, Kathryn A.; Tyson, Reed L.; Schneider, Tanya L.

2016-01-01

The LasR regulator protein functions at the top of the Pseudomonas aeruginosa quorum-sensing hierarchy and is implicated in promoting bacterial virulence. Of note is recent evidence that this transcription factor may also respond to oxidative stress. Here, all cysteines in LasR were inspected to deduce their redox sensitivity and to probe the connection between stress response and LasR activity using purified LasR and individual LasR domains. Cys79 in the ligand binding domain of LasR appears to be important for ligand recognition and folding of this domain to potentiate DNA binding but does not seem to be sensitive to oxidative stress when bound to its native ligand. Two cysteines in the DNA binding domain of LasR do form a disulfide bond when treated with hydrogen peroxide, and formation of this Cys201-Cys203 disulfide bond appears to disrupt the DNA binding activity of the transcription factor. Mutagenesis of either of these cysteines leads to expression of a protein that no longer binds DNA. A cell-based reporter assay linking LasR function with β-galactosidase activity gave results consistent with those obtained with purified LasR. This work provides a possible mechanism for oxidative stress response by LasR and indicates that multiple cysteines within the protein may prove to be useful targets for disabling its activity. PMID:27053110

Small Heat Shock Proteins in Redox Metabolism: Implications for Cardiovascular Diseases

PubMed Central

Christians, Elisabeth S.; Ishiwata, Takahiro; Benjamin, Ivor J.

2012-01-01

A timely review series on small heat shock proteins has to appropriately examine their fundamental properties and implications in the cardiovascular system since several members of this chaperone family exhibit robust expression in the myocardium and blood vessels. Due to energetic and metabolic demands, the cardiovascular system maintains a high mitochondrial activity but irreversible oxidative damage might ensue from increased production of reactive oxygen species. How equilibrium between their production and scavenging is achieved becomes paramount for physiological maintenance. For example, heat shock protein B1 (HSPB1) is implicated in maintaining this equilibrium or redox homeostasis by upholding the level of glutathione, a major redox mediator. Studies of gain or loss of function achieved by genetic manipulations have been highly informative for understanding the roles of those proteins. For example, genetic deficiency of several small heat shock proteins such as HSPB5 and HSPB2 is well-tolerated in heart cells whereas a single missense mutation causes human pathology. Such evidence highlights both the profound genetic redundancy observed among the multigene family of small heat shock proteins while underscoring the role proteotoxicity plays in driving disease pathogenesis. We will discuss the available data on small heat shock proteins in the cardiovascular system, redox metabolism and human diseases. From the medical perspective, we envision that such emerging knowledge of the multiple roles small heat shock proteins exert in the cardiovascular system will undoubtedly open new avenues for their identification and possible therapeutic targeting in humans. PMID:22710345

Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

PubMed

Ganini, Douglas; Leinisch, Fabian; Kumar, Ashutosh; Jiang, JinJie; Tokar, Erik J; Malone, Christine C; Petrovich, Robert M; Mason, Ronald P

2017-08-01

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H 2 O 2 ) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H 2 O 2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O 2 •- ) and H 2 O 2 in the presence of NADH. Generation of the free radical O 2 •- and H 2 O 2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies. Published by Elsevier B.V.

Oxidative/nitrosative stress and antidepressants: targets for novel antidepressants.

PubMed

Lee, Seung-Yup; Lee, Soo-Jung; Han, Changsu; Patkar, Ashwin A; Masand, Prakash S; Pae, Chi-Un

2013-10-01

The brain is an organ predisposed to oxidative/nitrosative stress. This is especially true in the case of aging as well as several neurodegenerative diseases. Under such circumstances, a decline in the normal antioxidant defense mechanisms leads to an increase in the vulnerability of the brain to the deleterious effects of oxidative damage. Highly reactive oxygen/nitrogen species damage lipids, proteins, and mitochondrial and neuronal genes. Unless antioxidant defenses react appropriately to damage inflicted by radicals, neurons may experience microalteration, microdysfunction, and degeneration. We reviewed how oxidative and nitrosative stresses contribute to the pathogenesis of depressive disorders and reviewed the clinical implications of various antioxidants as future targets for antidepressant treatment. Copyright © 2012 Elsevier Inc. All rights reserved.

Disruption of chaperone-mediated autophagy-dependent degradation of MEF2A by oxidative stress-induced lysosome destabilization

PubMed Central

Zhang, Li; Sun, Yang; Fei, Mingjian; Tan, Cheng; Wu, Jing; Zheng, Jie; Tang, Jiqing; Sun, Wei; Lv, Zhaoliang; Bao, Jiandong; Xu, Qiang; Yu, Huixin

2014-01-01

Oxidative stress has been implicated in both normal aging and various neurodegenerative disorders and it may be a major cause of neuronal death. Chaperone-mediated autophagy (CMA) targets selective cytoplasmic proteins for degradation by lysosomes and protects neurons against various extracellular stimuli including oxidative stress. MEF2A (myocyte enhancer factor 2A), a key transcription factor, protects primary neurons from oxidative stress-induced cell damage. However, the precise mechanisms of how the protein stability and the transcriptional activity of MEF2A are regulated under oxidative stress remain unknown. In this study, we report that MEF2A is physiologically degraded through the CMA pathway. In pathological conditions, mild oxidative stress (200 μM H2O2) enhances the degradation of MEF2A as well as its activity, whereas excessive oxidative stress (> 400 μM H2O2) disrupts its degradation process and leads to the accumulation of nonfunctional MEF2A. Under excessive oxidative stress, an N-terminal HDAC4 (histone deacetylase 4) cleavage product (HDAC4-NT), is significantly induced by lysosomal serine proteases released from ruptured lysosomes in a PRKACA (protein kinase, cAMP-dependent, catalytic, α)-independent manner. The production of HDAC4-NT, as a MEF2 repressor, may account for the reduced DNA-binding and transcriptional activity of MEF2A. Our work provides reliable evidence for the first time that MEF2A is targeted to lysosomes for CMA degradation; oxidative stress-induced lysosome destabilization leads to the disruption of MEF2A degradation as well as the dysregulation of its function. These findings may shed light on the underlying mechanisms of pathogenic processes of neuronal damage in various neurodegenerative-related diseases. PMID:24879151

The role of hypernitrosylation in the pathogenesis and pathophysiology of neuroprogressive diseases.

PubMed

Morris, Gerwyn; Walder, Ken; Carvalho, André F; Tye, Susannah J; Lucas, Kurt; Berk, Michael; Maes, Michael

2018-01-01

There is a wealth of data indicating that de novo protein S-nitrosylation in general and protein transnitrosylation in particular mediates the bulk of nitric oxide signalling. These processes enable redox sensing and facilitate homeostatic regulation of redox dependent protein signalling, function, stability and trafficking. Increased S-nitrosylation in an environment of increasing oxidative and nitrosative stress (O&NS) is initially a protective mechanism aimed at maintaining protein structure and function. When O&NS becomes severe, mechanisms governing denitrosylation and transnitrosylation break down leading to the pathological state referred to as hypernitrosylation (HN). Such a state has been implicated in the pathogenesis and pathophysiology of several neuropsychiatric and neurodegenerative diseases and we investigate its potential role in the development and maintenance of neuroprogressive disorders. In this paper, we propose a model whereby the hypernitrosylation of a range of functional proteins and enzymes lead to changes in activity which conspire to produce at least some of the core abnormalities contributing to the development and maintenance of pathology in these illnesses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hematopoietic stem cells burn fat to prevent exhaustion.

PubMed

Lallemand-Breitenbach, Valerie; de Thé, Hugues

2012-10-05

Ito et al. (2012) recently report in Nature Medicine that fatty acid oxidation (FAO) regulated by PPARδ controls asymmetric division in hematopoietic stem cells (HSCs). This metabolic mechanism prevents HSC exhaustion and is downstream of the promyelocytic leukemia protein PML, suggesting therapeutic implications for HSC function and disease. Copyright © 2012 Elsevier Inc. All rights reserved.

Oxidative inactivation of the endogenous antioxidant protein DJ-1 by the food contaminants 3-MCPD and 2-MCPD.

PubMed

Buhrke, Thorsten; Voss, Linn; Briese, Anja; Stephanowitz, Heike; Krause, Eberhard; Braeuning, Albert; Lampen, Alfonso

2018-01-01

3-Chloro-1,2-propanediol (3-MCPD) and 2-chloro-1,3-propanediol (2-MCPD) are heat-induced food contaminants being present either as free substances or as fatty acid esters in numerous foods. 3-MCPD was classified to be possibly carcinogenic to humans (category 2B) with kidney and testis being the primary target organs according to animal studies. A previous 28-day oral feeding study with rats revealed that the endogenous antioxidant protein DJ-1 was strongly deregulated at the protein level in kidney, liver, and testis of the experimental animals that had been treated either with 3-MCPD, 2-MCPD or their dipalmitate esters. Here we show that this deregulation is due to the oxidation of a conserved, redox-active cysteine residue (Cys106) of DJ-1 to a cysteine sulfonic acid which is equivalent to loss of function of DJ-1. Irreversible oxidation of DJ-1 is associated with a number of oxidative stress-related diseases such as Parkinson, cancer, and type II diabetes. It is assumed that 3-MCPD or 2-MCPD do not directly oxidize DJ-1, but that these substances induce the formation of reactive oxygen species (ROS) which in turn trigger DJ-1 oxidation. The implications of 3-MCPD/2-MCPD-mediated ROS formation in vivo for the ongoing risk assessment of these compounds as well as the potential of oxidized DJ-1 to serve as a novel effect biomarker for 3-MCPD/2-MCPD toxicity are being discussed.

Oxidation of Са2+-Binding Domain of NADPH Oxidase 5 (NOX5): Toward Understanding the Mechanism of Inactivation of NOX5 by ROS.

PubMed

Petrushanko, Irina Yu; Lobachev, Vladimir M; Kononikhin, Alexey S; Makarov, Alexander A; Devred, Francois; Kovacic, Hervé; Kubatiev, Aslan A; Tsvetkov, Philipp O

2016-01-01

NOX5 protein, one of the most active generators of reactive oxygen species (ROS), plays an important role in many processes, including regulation of cell growth, death and differentiation. Because of its central role in ROS generation, it needs to be tightly regulated to guarantee cellular homeostasis. Contrary to other members of NADPH-oxidases family, NOX5 has its own regulatory calcium-binding domain and thus could be activated directly by calcium ions. While several mechanisms of activation have been described, very little is known about the mechanisms that could prevent the overproduction of ROS by NOX5. In the present study using calorimetric methods and circular dichroism we found that oxidation of cysteine and methionine residues of NOX5 decreases binding of Ca2+ ions and perturbs both secondary and tertiary structure of protein. Our data strongly suggest that oxidation of calcium-binding domain of NOX5 could be implicated in its inactivation, serving as a possible defense mechanism against oxidative stress.

Transglutaminase is a therapeutic target for oxidative stress, excitotoxicity and stroke: a new epigenetic kid on the CNS block

PubMed Central

Basso, Manuela; Ratan, Rajiv R

2013-01-01

Transglutaminases (TGs) are multifunctional, calcium-dependent enzymes that have been recently implicated in stroke pathophysiology. Classically, these enzymes are thought to participate in cell injury and death in chronic neurodegenerative conditions via their ability to catalyze covalent, nondegradable crosslinks between proteins or to incorporate polyamines into protein substrates. Accumulating lines of inquiry indicate that specific TG isoforms can shuttle into the nucleus when they sense pathologic changes in calcium or oxidative stress, bind to chromatin and thereby transduce these changes into transcriptional repression of genes involved in metabolic or oxidant adaptation. Here, we review the evidence that supports principally a role for one isoform of this family, TG2, in cell injury and death associated with hemorrhagic or ischemic stroke. We also outline an evolving model in which TG2 is a critical mediator between pathologic signaling and epigenetic modifications that lead to gene repression. Accordingly, the salutary effects of TG inhibitors in stroke may derive from their ability to restore homeostasis by removing inappropriate deactivation of adaptive genetic programs by oxidative stress or extrasynaptic glutamate receptor signaling. PMID:23571278

Thiamine deficiency induces oxidative stress and exacerbates the plaque pathology in Alzheimer’s mouse model

PubMed Central

Karuppagounder, Saravanan S.; Xu, Hui; Shi, Qingli; Chen, Lian H.; Pedrini, Steve; Pechman, David; Baker, Harriet; Beal, M. Flint; Gandy, Sam E.; Gibson, Gary E.

2009-01-01

Mitochondrial dysfunction, oxidative stress and reductions in thiamine-dependent enzymes have been implicated in multiple neurological disorders including Alzheimer's disease (AD). Experimental thiamine deficiency (TD) is an established model for reducing the activities of thiamine-dependent enzymes in brain. TD diminishes thiamine dependent enzymes throughout the brain, but produces a time-dependent selective neuronal loss, glial activation, inflammation, abnormalities in oxidative metabolism and clusters of degenerating neurites in only specific thalamic regions. The present studies tested how TD alters brain pathology in Tg19959 transgenic mice over expressing a double mutant form of the amyloid precursor protein (APP). TD exacerbated amyloid plaque pathology in transgenic mice and enlarged the area occupied by plaques in cortex, hippocampus and thalamus by 50%, 200% and 200%, respectively. TD increased Aβ1–42 levels by about three-fold, β-CTF (C99) levels by 33% and β-secretase (BACE1) protein levels by 43%. TD induced inflammation in areas of plaque formation. Thus, the induction of mild impairment of oxidative metabolism, oxidative stress and inflammation induced by TD alters metabolism of APP and/or Aβ and promotes accumulation of plaques independent of neuron loss or neuritic clusters. PMID:18406011

Nitric oxide and heat shock protein 90 co-regulate temperature-induced bleaching in the soft coral Eunicea fusca

NASA Astrophysics Data System (ADS)

Ross, Cliff

2014-06-01

Coral bleaching represents a complex physiological process that is affected not only by environmental conditions but by the dynamic internal cellular biology of symbiotic dinoflagellates ( Symbiodinium spp.) and their cnidarian hosts. Recently, nitric oxide (NO) has emerged as a key molecule involved with the expulsion of Symbiodinium from host cnidarian cells. However, the site of production remains under debate, and the corresponding signaling pathways within and between host and endosymbiont remain elusive. In this study, using freshly isolated Symbiodinium from the soft coral Eunicea fusca, I demonstrate that thermally induced stress causes an upregulation in Symbiodinium heat shock protein 90 (Hsp90). In turn, Hsp90 shows a concomitant ability to enhance the activity of a constitutively expressed isoform of NO synthase. The resulting production of NO constitutes a signaling molecule capable of inducing Symbiodinium expulsion. Using nitric oxide synthase (NOS) and Hsp90 polyclonal antibodies, thermal stress-induced Hsp90 was shown to co-immunoprecipitate with a constitutive isoform of NOS. The specific blocking of Hsp90 activity, with the Hsp90 inhibitor geldanamycin, was capable of inhibiting NO production implicating the involvement of a coordinated regulatory system. These results have strong evolutionary implications for Hsp90-NOS chaperone complexes among biological kingdoms and provide evidence for a new functional role in symbiotic associations.

Immuno-spin trapping of heme-induced protein radicals: Implications for heme oxygenase-1 induction and heme degradation

PubMed Central

Ganini, Douglas; Deterding, Leesa J.; Ehrenshaft, Marilyn; Chatterjee, Saurabh; Mason, Ronald P.

2013-01-01

Heme, in the presence of hydrogen peroxide, can act as a peroxidase. Intravascular hemolysis results in a massive release of heme into the plasma in several pathophysiological conditions such as hemolytic anemia, malaria, and sickle cell disease. Heme is known to induce heme oxygenase-1(HO-1) expression, and the extent of induction depends on the ratio of albumin to heme in plasma. HO-1 degrades heme and ultimately generates the antioxidant bilirubin. Heme also causes oxidative stress in cells, but whether it causes protein-radical formation has not yet been studied. In the literature, two purposes for the degradation of heme by HO-1 are discussed. One is the production of the antioxidant bilirubin and the other is the prevention of heme-dependent adverse effects. Here we have investigated heme-induced protein-radical formation, which might have pathophysiological consequences, and have used immunospin trapping to establish the formation of heme-induced protein radicals in two systems: human serum albumin (HSA)/H2O2 and human plasma/H2O2.We found that excess heme catalyzed the formation of HSA radicals in the presence of hydrogen peroxide. When heme and hydrogen peroxide were added to human plasma, heme was found to oxidize proteins, primarily and predominantly HSA; however, when HSA-depleted plasma was used, heme triggered the oxidation of several other proteins, including transferrin. Thus, HSA in plasma protected other proteins from heme/H2O2-induced oxidation. The antioxidants ascorbate and uric acid significantly attenuated protein-radical formation induced by heme/ H2O2; however, bilirubin did not confer significant protection. Based on these findings, we conclude that heme is degraded by HO-1 because it is a catalyst of protein-radical formation and not merely to produce the relatively inefficient antioxidant bilirubin. PMID:23624303

G protein-coupled estrogen receptor (GPER) deficiency induces cardiac remodeling through oxidative stress.

PubMed

Wang, Hao; Sun, Xuming; Lin, Marina S; Ferrario, Carlos M; Van Remmen, Holly; Groban, Leanne

2018-04-25

Oxidative stress has been implicated in the unfavorable changes in cardiac function and remodeling that occur after ovarian estrogen loss. Using ovariectomized rat models, we previously reported that the cardioprotective actions of estrogen are mediated by the G protein-coupled estrogen receptor (GPER). Here, in 9-month-old, female cardiomyocyte-specific GPER knockout (KO) mice vs sex- and age-matched wild-type (WT) mice, we found increased cardiac oxidative stress and oxidant damage, measured as a decreased ratio of reduced glutathione to oxidized glutathione, increased 4-hydroxynonenal and 8-hydroxy-2'-deoxyguanosine (8-oxo-DG) staining, and increased expression of oxidative stress-related genes. GPER KO mice also displayed increased heart weight, cardiac collagen deposition, and Doppler-derived filling pressure, and decreased percent fractional shortening and early mitral annular velocity compared with WT controls. Treatment of GPER KO mice for 8 weeks with phosphonium [10-(4,5-dimethoxy-2-methyl 3,6-dioxo-1,4-cyclohexadien-1-yl)decyl] triphenyl-,mesylate (MitoQ), a mitochondria-targeted antioxidant, significantly attenuated these measures of cardiac dysfunction, and MitoQ decreased 8-oxo-DG intensity compared with treatment with an inactive comparator compound, (1-decyl)triphenylphosphonium bromide (P <0.05). A real-time polymerase chain reaction array analysis of 84 oxidative stress and antioxidant defense genes revealed that MitoQ attenuates the increase in NADPH oxidase 4 and prostaglandin-endoperoxide synthase 2 and the decrease in uncoupling protein 3 and glutathione S-transferase kappa 1 seen in GPER KO mice. Our findings suggest that the cardioprotective effects of GPER include an antioxidant role and that targeted strategies to limit oxidative stress after early noncancerous surgical extirpation of ovaries or menopause may help limit alterations in cardiac structure and function related to estrogen loss. Copyright © 2018 Elsevier Inc. All rights reserved.

DJ-1 Is a Copper Chaperone Acting on SOD1 Activation*

PubMed Central

Girotto, Stefania; Cendron, Laura; Bisaglia, Marco; Tessari, Isabella; Mammi, Stefano; Zanotti, Giuseppe; Bubacco, Luigi

2014-01-01

Lack of oxidative stress control is a common and often prime feature observed in many neurodegenerative diseases. Both DJ-1 and SOD1, proteins involved in familial Parkinson disease and amyotrophic lateral sclerosis, respectively, play a protective role against oxidative stress. Impaired activity and modified expression of both proteins have been observed in different neurodegenerative diseases. A potential cooperative action of DJ-1 and SOD1 in the same oxidative stress response pathway may be suggested based on a copper-mediated interaction between the two proteins reported here. To investigate the mechanisms underlying the antioxidative function of DJ-1 in relation to SOD1 activity, we investigated the ability of DJ-1 to bind copper ions. We structurally characterized a novel copper binding site involving Cys-106, and we investigated, using different techniques, the kinetics of DJ-1 binding to copper ions. The copper transfer between the two proteins was also examined using both fluorescence spectroscopy and specific biochemical assays for SOD1 activity. The structural and functional analysis of the novel DJ-1 copper binding site led us to identify a putative role for DJ-1 as a copper chaperone. Alteration of the coordination geometry of the copper ion in DJ-1 may be correlated to the physiological role of the protein, to a potential failure in metal transfer to SOD1, and to successive implications in neurodegenerative etiopathogenesis. PMID:24567322

LDL oxidation by THP-1 monocytes: implication of HNP-1, SgIII and DMT-1.

PubMed

He, Chunyan; Huang, Rui; Du, Fen; Zheng, Fang; Wei, Lei; Wu, Junzhu

2009-04-01

Oxidized low-density lipoprotein (oxLDL) plays an important role in the pathogenesis of atherosclerosis. However, the mechanisms of the initiation and progression of LDL oxidation by cells are still unknown. We investigated the molecular mechanism underlying THP-1 cell-mediated LDL oxidation. LDL oxidation was monitored at 234 nm by detecting the formation of conjugated dienes. cDNA array analysis was applied to profile changes in gene expression of human THP-1 monocytes in response to LDL stimulation. The mRNA and protein levels of secretogranin III (SgIII), divalent metal transporter (DMT-1) and human alpha-defensin 1 (HNP-1) were determined by real-time RT-PCR and Western blotting respectively. Eukaryotic expression vectors containing full-length cDNA sequence of HNP-1 (pEGFP-C1/HNP-1) SgIII (pEGFP-C1/SgIII) or DMT-1 (pEGFP-C1/DMT-1) were constructed and transfected to THP-1 cells. The effects of overexpression of these three genes on THP-1 cell-mediated LDL oxidation were observed. LDL oxidation was most pronounced after LDL was incubated with THP-1 cells for 9 h. 1651 genes in total were detected by cDNA array analysis in THP-1 cells with or without LDL treatment for 9 h. Thirteen genes with >2-fold relative expression difference were identified, including nine genes whose expression was up-regulated and four genes whose expression was down-regulated. Among the up-regulated genes, SgIII, DMT-1 and HNP-1 were reported to be associated with atherosclerosis. The increased mRNA expressions of these three genes were confirmed by real-time RT-PCR. Western blotting analysis demonstrated that protein expressions of SgIII and DMT-1 were also enhanced in THP-1 cells in response to LDL. Furthermore, transient overexpression of HNP-1, SgIII or DMT-1 in THP-1 cells significantly increased THP-1 cell-mediated LDL oxidation. Our data suggest that SgIII, DMT-1 and HNP-1 are implicated in cell-mediated LDL oxidation.

Ebselen alters cellular oxidative status and induces endoplasmic reticulum stress in rat hippocampal astrocytes.

PubMed

Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; de la Casa-Resino, Irene; Martinez-Ruiz, Antonio; Perez-Lopez, Marcos; Portilla, Juan C; Salido, Gines M; Gonzalez, Antonio

2016-05-16

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. Because of its properties, it may be protective against injury to the nervous tissue. However, evidence suggests that its glutathione peroxidase activity could underlie certain deleterious actions on cell physiology. In this study we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular oxidative status, cytosolic free-Ca(2+) concentration ([Ca(2+)]c), setting of endoplasmic reticulum stress and phosphorylation of glial fibrillary acidic protein and major mitogen-activated protein kinases were analyzed. Our results show that ebselen induced a concentration-dependent increase in the generation of reactive oxygen species in the mitochondria. We observed a concentration-dependent increase in global cysteine oxidation and in the level of malondialdehyde in the presence of ebselen. We also detected increases in catalase, glutathione S-transferase and glutathione reductase activity. Ebselen also evoked a concentration-dependent increase in [Ca(2+)]c. Moreover, we observed a concentration-dependent increase in the phosphorylation of the unfolded protein response markers, eukaryotic translation initiation factor 2α and X-box binding protein 1. Finally, ebselen also induced an increase in the phosphorylation of glial fibrillary acidic protein, SAPK/JNK, p38 MAPK and p44/42 MAPK. Our results provide strong evidence that implicate endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in an oxidative damage of cells in the presence of ebselen. The compound thus might exert deleterious actions on astrocyte physiology that could compromise their function. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Tyrosine oxidation in heme oxygenase: examination of long-range proton-coupled electron transfer.

PubMed

Smirnov, Valeriy V; Roth, Justine P

2014-10-01

Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX (Por) to biliverdin, CO, and the free ferrous ion. Described here are studies of tyrosyl radical formation reactions that occur after oxidizing Fe(III)(Por) to Fe(IV)=O(Por(·+)) in human heme oxygenase isoform-1 (hHO-1) and the structurally homologous protein from Corynebacterium diphtheriae (cdHO). Site-directed mutagenesis on hHO-1 probes the reduction of Fe(IV)=O(Por(·+)) by tyrosine residues within 11 Å of the prosthetic group. In hHO-1, Y58· is implicated as the most likely site of oxidation, based on the pH and pD dependent kinetics. The absence of solvent deuterium isotope effects in basic solutions of hHO-1 and cdHO contrasts with the behavior of these proteins in the acidic solution, suggesting that long-range proton-coupled electron transfer predominates over electron transfer.

Essential role of FBXL5-mediated cellular iron homeostasis in maintenance of hematopoietic stem cells

PubMed Central

Muto, Yoshiharu; Nishiyama, Masaaki; Nita, Akihiro; Moroishi, Toshiro; Nakayama, Keiichi I.

2017-01-01

Hematopoietic stem cells (HSCs) are maintained in a hypoxic niche to limit oxidative stress. Although iron elicits oxidative stress, the importance of iron homeostasis in HSCs has been unknown. Here we show that iron regulation by the F-box protein FBXL5 is required for HSC self-renewal. Conditional deletion of Fbxl5 in mouse HSCs results in cellular iron overload and a reduced cell number. Bone marrow transplantation reveals that FBXL5-deficient HSCs are unable to reconstitute the hematopoietic system of irradiated recipients as a result of stem cell exhaustion. Transcriptomic analysis shows abnormal activation of oxidative stress responses and the cell cycle in FBXL5-deficient mouse HSCs as well as downregulation of FBXL5 expression in HSCs of patients with myelodysplastic syndrome. Suppression of iron regulatory protein 2 (IRP2) accumulation in FBXL5-deficient mouse HSCs restores stem cell function, implicating IRP2 as a potential therapeutic target for human hematopoietic diseases associated with FBXL5 downregulation. PMID:28714470

Generator-specific targets of mitochondrial reactive oxygen species.

PubMed

Bleier, Lea; Wittig, Ilka; Heide, Heinrich; Steger, Mirco; Brandt, Ulrich; Dröse, Stefan

2015-01-01

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for "signaling" and "damaging" ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc1 complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H2O2) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of Single and Combined Losartan and Tempol Treatments on Oxidative Stress, Kidney Structure and Function in Spontaneously Hypertensive Rats with Early Course of Proteinuric Nephropathy

PubMed Central

Grujic-Milanovic, Jelica; Miloradovic, Zoran; Ivanov, Milan; Jovovic, Djurdjica; Vajic, Una-Jovana; Zivotic, Maja; Markovic-Lipkovski, Jasmina; Mihailovic-Stanojevic, Nevena

2016-01-01

Oxidative stress has been widely implicated in both hypertension and chronic kidney disease (CKD). Hypertension is a major risk factor for CKD progression. In the present study we have investigated the effects of chronic single tempol (membrane-permeable radical scavenger) or losartan (angiotensin II type 1 receptor blocker) treatment, and their combination on systemic oxidative status (plasma thiobarbituric acid-reactive substances (pTBARS) production, plasma antioxidant capacity (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, pABTS), erythrocyte antioxidant enzymes activities) and kidney oxidative stress (kTBARS, kABTS, kidney antioxidant enzymes activities), kidney function and structure in spontaneously hypertensive rats (SHR) with the early course of adriamycin-induced nephropathy. Adult SHR were divided into five groups. The control group received vehicle, while the other groups received adriamycin (2 mg/kg, i.v.) twice in a 21-day interval, followed by vehicle, losartan (L,10 mg/kg/day), tempol (T,100 mg/kg/day) or combined T+L treatment (by gavage) during a six-week period. Adriamycin significantly increased proteinuria, plasma lipid peroxidation, kidney protein oxidation, nitrite excretion, matrix metalloproteinase-1 (MMP-1) protein expression and nestin immunostaining in the kidney. Also, it decreased kidney antioxidant defense, kidney NADPH oxidase 4 (kNox4) protein expression and abolished anti-inflammatory response due to significant reduction of kidney NADPH oxidase 2 (kNox2) protein expression in SHR. All treatments reduced protein-to-creatinine ratio (marker of proteinuria), pTBARS production, kidney protein carbonylation, nitrite excretion, increased antioxidant capacity and restored kidney nestin expression similar to control. Both single treatments significantly improved systemic and kidney antioxidant defense, bioavailability of renal nitric oxide, reduced kMMP-1 protein expression and renal injury, thus retarded CKD progression. Losartan improved blood pressure, as well as tubular injury and restored anti-inflammatory defense by reverting kNox2 expression to the control level. Interestingly, tempol was more successful in reducing systemic oxidative stress, proteinuria, kMMP-1 and glomerulosclerosis. However, combined treatment failed to overcome the beneficial effects of single treatments in slowing down the progression of ADR-induced nephropathy in SHR. PMID:27560781

Expression and Activity of Nitric Oxide Synthase Isoforms in Methamphetamine-Induced Striatal Dopamine Toxicity

PubMed Central

Friend, Danielle M.; Son, Jong H.; Keefe, Kristen A.

2013-01-01

Nitric oxide is implicated in methamphetamine (METH)-induced neurotoxicity; however, the source of the nitric oxide has not been identified. Previous work has also revealed that animals with partial dopamine loss induced by a neurotoxic regimen of methamphetamine fail to exhibit further decreases in striatal dopamine when re-exposed to methamphetamine 7–30 days later. The current study examined nitric oxide synthase expression and activity and protein nitration in striata of animals administered saline or neurotoxic regimens of methamphetamine at postnatal days 60 and/or 90, resulting in four treatment groups: Saline:Saline, METH:Saline, Saline:METH, and METH:METH. Acute administration of methamphetamine on postnatal day 90 (Saline:METH and METH:METH) increased nitric oxide production, as evidenced by increased protein nitration. Methamphetamine did not, however, change the expression of endothelial or inducible isoforms of nitric oxide synthase, nor did it change the number of cells positive for neuronal nitric oxide synthase mRNA expression or the amount of neuronal nitric oxide synthase mRNA per cell. However, nitric oxide synthase activity in striatal interneurons was increased in the Saline:METH and METH:METH animals. These data suggest that increased nitric oxide production after a neurotoxic regimen of methamphetamine results from increased nitric oxide synthase activity, rather than an induction of mRNA, and that constitutively expressed neuronal nitric oxide synthase is the most likely source of nitric oxide after methamphetamine administration. Of interest, animals rendered resistant to further methamphetamine-induced dopamine depletions still show equivalent degrees of methamphetamine-induced nitric oxide production, suggesting that nitric oxide production alone in response to methamphetamine is not sufficient to induce acute neurotoxic injury. PMID:23230214

High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis.

PubMed Central

Nagaraj, R H; Sell, D R; Prabhakaram, M; Ortwerth, B J; Monnier, V M

1991-01-01

Pentosidine is a recently discovered protein crosslink, involving lysine and arginine residues linked together in an imidazo [4,5,6] pyridinium ring formed by a 5-carbon sugar during nonenzymatic browning (Maillard reaction). The presence of high ascorbate levels in the human lens and its ability to undergo nonenzymatic browning led us to investigate pentosidine formation in the aging human lens. Incubation of lens crystallins with ascorbate and its oxidation products dehydroascorbate and 2,3-diketogulonate leads progressively to the formation of pentosidine crosslinks in the presence of oxygen. Under nitrogen, however, pentosidine forms only from 2,3-diketogulonate or xylosone, a degradation product of 2,3-diketogulonate. A high correlation between pentosidine crosslinks and the degree of lens pigmentation is noted in cataractous lenses. Pentosidine is found to be primarily associated with alpha-crystallin fractions of 300-5000 kDa. These results suggest that redox imbalance in cellular senescent systems such as the ocular lens may lead to irreversible ascorbate oxidation and protein crosslinking by xylosone. This mechanism may play an important role in the pathogenesis of "brunescent" cataracts. Images PMID:1946446

Effectiveness of exercise and protein supplementation intervention on body composition, functional fitness, and oxidative stress among elderly Malays with sarcopenia.

PubMed

Shahar, Suzana; Kamaruddin, Norshafarina Shari; Badrasawi, Manal; Sakian, Noor Ibrahim Mohamed; Abd Manaf, Zahara; Yassin, Zaitun; Joseph, Leonard

2013-01-01

Sarcopenia, characterized as muscle loss that occurs with aging, is a major health problem in an aging population, due to its implications on mobility, quality of life, and fall risk. Protein supplementation could improve the physical fitness by increasing protein anabolism, and exercise has a documented evidence of positive effect on functional status among the elderly. However, the combined effect of both protein supplementation and exercise has not been investigated among sarcopenic elderly in the Asian population. Thus, this study aimed to determine the effectiveness of exercise intervention and protein supplementation either alone or in combination for 12 weeks, on body composition, functional fitness, and oxidative stress among elderly Malays with sarcopenia. Sixty five sarcopenic elderly Malays aged 60-74 years were assigned to the control group, exercise group (ExG), protein supplementation group (PrG), or the combination of exercise and protein supplementation group. A significant interaction effect between body weight and body mass index (BMI) was observed, with the PrG (-2.1% body weight, -1.8% BMI) showing the highest reductions. Further, there was a decrease in % body fat (-4.5%) and an increase in fat-free mass (kg) (+5.7%) in the ExG after 12 weeks (P < 0.05). The highest increments in lower and upper body strength were observed in the PrG (73.2%) and ExG (47.6%), respectively. In addition, the ExG showed a reduction in superoxide dismutase (SOD) levels, and both interventions did not alter either lipid or protein oxidation. In conclusion, the exercise program was found to improve muscle strength and body composition, while protein supplementation reduced body weight and increased upper body strength, among sarcopenic elderly in Malaysia.

Effectiveness of exercise and protein supplementation intervention on body composition, functional fitness, and oxidative stress among elderly Malays with sarcopenia

PubMed Central

Shahar, Suzana; Kamaruddin, Norshafarina Shari; Badrasawi, Manal; Sakian, Noor Ibrahim Mohamed; Manaf, Zahara Abd; Yassin, Zaitun; Joseph, Leonard

2013-01-01

Sarcopenia, characterized as muscle loss that occurs with aging, is a major health problem in an aging population, due to its implications on mobility, quality of life, and fall risk. Protein supplementation could improve the physical fitness by increasing protein anabolism, and exercise has a documented evidence of positive effect on functional status among the elderly. However, the combined effect of both protein supplementation and exercise has not been investigated among sarcopenic elderly in the Asian population. Thus, this study aimed to determine the effectiveness of exercise intervention and protein supplementation either alone or in combination for 12 weeks, on body composition, functional fitness, and oxidative stress among elderly Malays with sarcopenia. Sixty five sarcopenic elderly Malays aged 60–74 years were assigned to the control group, exercise group (ExG), protein supplementation group (PrG), or the combination of exercise and protein supplementation group. A significant interaction effect between body weight and body mass index (BMI) was observed, with the PrG (−2.1% body weight, −1.8% BMI) showing the highest reductions. Further, there was a decrease in % body fat (−4.5%) and an increase in fat-free mass (kg) (+5.7%) in the ExG after 12 weeks (P < 0.05). The highest increments in lower and upper body strength were observed in the PrG (73.2%) and ExG (47.6%), respectively. In addition, the ExG showed a reduction in superoxide dismutase (SOD) levels, and both interventions did not alter either lipid or protein oxidation. In conclusion, the exercise program was found to improve muscle strength and body composition, while protein supplementation reduced body weight and increased upper body strength, among sarcopenic elderly in Malaysia. PMID:24143082

Cell signaling by reactive nitrogen and oxygen species in atherosclerosis

NASA Technical Reports Server (NTRS)

Patel, R. P.; Moellering, D.; Murphy-Ullrich, J.; Jo, H.; Beckman, J. S.; Darley-Usmar, V. M.

2000-01-01

The production of reactive oxygen and nitrogen species has been implicated in atherosclerosis principally as means of damaging low-density lipoprotein that in turn initiates the accumulation of cholesterol in macrophages. The diversity of novel oxidative modifications to lipids and proteins recently identified in atherosclerotic lesions has revealed surprising complexity in the mechanisms of oxidative damage and their potential role in atherosclerosis. Oxidative or nitrosative stress does not completely consume intracellular antioxidants leading to cell death as previously thought. Rather, oxidative and nitrosative stress have a more subtle impact on the atherogenic process by modulating intracellular signaling pathways in vascular tissues to affect inflammatory cell adhesion, migration, proliferation, and differentiation. Furthermore, cellular responses can affect the production of nitric oxide, which in turn can strongly influence the nature of oxidative modifications occurring in atherosclerosis. The dynamic interactions between endogenous low concentrations of oxidants or reactive nitrogen species with intracellular signaling pathways may have a general role in processes affecting wound healing to apoptosis, which can provide novel insights into the pathogenesis of atherosclerosis.

Decoding telomere protein Rap1: Its telomeric and nontelomeric functions and potential implications in diabetic cardiomyopathy.

PubMed

Cai, Yin; Kandula, Vidya; Kosuru, Ramoji; Ye, Xiaodong; Irwin, Michael G; Xia, Zhengyuan

2017-10-02

Mammalian Rap1, the most conserved telomere-interacting protein, beyond its role within nucleus for the maintenance of telomeric functions, is also well known for its pleiotropic functions in various physiological and pathological conditions associated with metabolism, inflammation and oxidative stress. For all these, nowadays Rap1 is the subject of critical investigations aimed to unveil its molecular signaling pathways and to scrutinize the applicability of its modulation as a promising therapeutic strategy with clinical relevance. However, the underlying intimate mechanisms of Rap1 are not extensively studied, but any modulation of this protein level has been associated with pathologies like inflammation, oxidative stress and deregulated metabolism. This is considerably important in light of the recent discovery of Rap1 modulation in diseases like cancer and cardiac metabolic disorders. In this review, we focus on both the telomeric and nontelomeric functions of Rap1 and its modulation in various health risks, especially on the heart.

Mitochondrial iron-sulfur cluster biogenesis from molecular understanding to clinical disease

PubMed Central

Alfadhel, Majid; Nashabat, Marwan; Ali, Qais Abu; Hundallah, Khalid

2017-01-01

Iron–sulfur clusters (ISCs) are known to play a major role in various protein functions. Located in the mitochondria, cytosol, endoplasmic reticulum and nucleus, they contribute to various core cellular functions. Until recently, only a few human diseases related to mitochondrial ISC biogenesis defects have been described. Such diseases include Friedreich ataxia, combined oxidative phosphorylation deficiency 19, infantile complex II/III deficiency defect, hereditary myopathy with lactic acidosis and mitochondrial muscle myopathy, lipoic acid biosynthesis defects, multiple mitochondrial dysfunctions syndromes and non ketotic hyperglycinemia due to glutaredoxin 5 gene defect. Disorders of mitochondrial import, export and translation, including sideroblastic anemia with ataxia, EVEN-PLUS syndrome and mitochondrial complex I deficiency due to nucleotide-binding protein-like protein gene defect, have also been implicated in ISC biogenesis defects. With advances in next generation sequencing technologies, more disorders related to ISC biogenesis defects are expected to be elucidated. In this article, we aim to shed the light on mitochondrial ISC biogenesis, related proteins and their function, pathophysiology, clinical phenotypes of related disorders, diagnostic approach, and future implications. PMID:28064324

Protein oxidative damage and heme oxygenase in sunlight-exposed human skin: roles of MAPK responses to oxidative stress.

PubMed

Akasaka, Emiko; Takekoshi, Susumu; Horikoshi, Yosuke; Toriumi, Kentarou; Ikoma, Norihiro; Mabuchi, Tomotaka; Tamiya, Shiho; Matsuyama, Takashi; Ozawa, Akira

2010-12-20

Oxidative stress derived from ultraviolet (UV) light in sunlight induces different hazardous effects in the skin, including sunburn, photo-aging and DNA mutagenesis. In this study, the protein-bound lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8OHdG) were investigated in chronically sun-exposed and sun-protected human skins using immunohistochemistry. The levels of antioxidative enzymes, such as heme oxygenase 1 and 2, Cu/Zn-SOD, Mn-SOD and catalase, were also examined. Oxidative stress is also implicated in the activation of signal transduction pathways, such as mitogen-activated protein kinase (MAPK). Therefore, the expression and distribution of phosphorylated p38 MAPK, phosphorylated Jun N-terminal kinase (JNK) and phosphorylated extracellular signal-regulated kinase (ERK) were observed. Skin specimens were obtained from the surgical margins. Chronically sunlight-exposed skin samples were taken from the ante-auricular (n = 10) and sunlight-protected skin samples were taken from the post-auricular (n = 10). HNE was increased in the chronically sunlight-exposed skin but not in the sunlight-protected skin. The expression of heme oxygenase-2 was markedly increased in the sunlight-exposed skin compared with the sun-protected skin. In contrast, the intensity of immunostaining of Cu/Zn-SOD, Mn-SOD and catalase was not different between the two areas. Phosphorylated p38 MAPK and phosphorylated JNK accumulated in the ante-auricular dermis and epidermis, respectively. These data show that particular anti-oxidative enzymes function as protective factors in chronically sunlight-exposed human skin. Taken together, our results suggest (1) antioxidative effects of heme oxygenase-2 in chronically sunlight-exposed human skin, and that (2) activation of p38 MAPK may be responsible for oxidative stress.

Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones.

PubMed

Srinivasan, Usha; Bala, Aveenash; Jao, Shu-chuan; Starke, David W; Jordan, T William; Mieyal, John J

2006-07-25

Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.

Fine Tuning of Redox Networks on Multiheme Cytochromes from Geobacter sulfurreducens Drives Physiological Electron/Proton Energy Transduction

PubMed Central

Morgado, Leonor; Dantas, Joana M.; Bruix, Marta; Londer, Yuri Y.; Salgueiro, Carlos A.

2012-01-01

The bacterium Geobacter sulfurreducens (Gs) can grow in the presence of extracellular terminal acceptors, a property that is currently explored to harvest electricity from aquatic sediments and waste organic matter into microbial fuel cells. A family composed of five triheme cytochromes (PpcA-E) was identified in Gs. These cytochromes play a crucial role by bridging the electron transfer from oxidation of cytoplasmic donors to the cell exterior and assisting the reduction of extracellular terminal acceptors. The detailed thermodynamic characterization of such proteins showed that PpcA and PpcD have an important redox-Bohr effect that might implicate these proteins in the e−/H+ coupling mechanisms to sustain cellular growth. The physiological relevance of the redox-Bohr effect in these proteins was studied by determining the fractional contribution of each individual redox-microstate at different pH values. For both proteins, oxidation progresses from a particular protonated microstate to a particular deprotonated one, over specific pH ranges. The preferred e−/H+ transfer pathway established by the selected microstates indicates that both proteins are functionally designed to couple e−/H+ transfer at the physiological pH range for cellular growth. PMID:22899897

Quercetin prevents protein nitration and glycolytic block of proliferation in hydrogen peroxide insulted cultured neuronal precursor cells (NPCs): Implications on CNS regeneration.

PubMed

Sajad, Mir; Zargan, Jamil; Zargar, Mohammad Afzal; Sharma, Jyoti; Umar, Sadiq; Arora, Rajesh; Khan, Haider A

2013-05-01

Survival along with optimal proliferation of neuronal precursors determines the outcomes of the endogenous cellular repair in CNS. Cellular-oxidation based cell death has been described in several neurodegenerative disorders. Therefore, this study was aimed at the identification of the potent targets of oxidative damage to the neuronal precursors and its effective prevention by a natural flavonoid, Quercetin. Neuronal precursor cells (NPCs), Nestin+ and GFAP (Glial fibrillary acidic protein)+ were isolated and cultured from adult rat SVZ (subventricular zone). These cells were challenged with a single dose of H2O2 (50μM) and/or pre-treated with different concentrations of Quercetin. H2O2 severely limited the cellular viability and expansion of the neurospheres. Cellular-oxidation studies revealed reduction in glutathione dependent redox buffering along with depletion of enzymatic cellular antioxidants that might potentiate the nitrite (NO2(-)) and superoxide anion (O2(-)) mediated peroxynitrite (ONOO(-)) formation and irreversible protein nitration. We identified depleted PK-M2 (M2 isoform of pyruvate kinase) activity and apoptosis of NPCs revealed by the genomic DNA fragmentation and elevated PARP (poly ADP ribose polymerase) activity along with increased Caspase activity initiated by severely depolarised mitochondrial membranes. However, the pre-treatment of Quercetin in a dose-response manner prevented these changes and restored the expansion of neurospheres preferably by neutralizing the oxidative conditions and thereby reducing peroxynitrite formation, protein nitration and PK-M2 depletion. Our results unravel the potential interactions of oxidative environment and respiration in the survival and activation of precursors and offer a promise shown by a natural flavonoid in the protective strategy for neuronal precursors of adult brain. Copyright © 2013 Elsevier Inc. All rights reserved.

Decrease in skin collagen glycation with improved glycemic control in patients with insulin-dependent diabetes mellitus.

PubMed Central

Lyons, T J; Bailie, K E; Dyer, D G; Dunn, J A; Baynes, J W

1991-01-01

Glycation, oxidation, and nonenzymatic browning of protein have all been implicated in the development of diabetic complications. The initial product of glycation of protein, fructoselysine (FL), undergoes further reactions, yielding a complex mixture of browning products, including the fluorescent lysine-arginine cross-link, pentosidine. Alternatively, FL may be cleaved oxidatively to form N(epsilon)-(carboxymethyl)lysine (CML), while glycated hydroxylysine, an amino-acid unique to collagen, may yield N(epsilon)-(carboxymethyl)hydroxylysine (CMhL). We have measured FL, pentosidine, fluorescence (excitation = 328 nm, emission = 378 nm), CML, and CMhL in insoluble skin collagen from 14 insulin-dependent diabetic patients before and after a 4-mo period of intensive therapy to improve glycemic control. Mean home blood glucose fell from 8.7 +/- 2.5 (mean +/- 1 SD) to 6.8 +/- 1.4 mM (P less than 0.005), and mean glycated hemoglobin (HbA1) from 11.6 +/- 2.3% to 8.3 +/- 1.1% (P less than 0.001). These changes were accompanied by a significant decrease in glycation of skin collagen, from 13.2 +/- 4.3 to 10.6 +/- 2.3 mmol FL/mol lysine (P less than 0.002). However, levels of browning and oxidation products (pentosidine, CML, and CMhL) and fluorescence were unchanged. These results show that the glycation of long-lived proteins can be decreased by improved glycemic control, but suggest that once cumulative damage to collagen by browning and oxidation reactions has occurred, it may not be readily reversed. Thus, in diabetic patients, institution and maintenance of good glycemic control at any time could potentially limit the extent of subsequent long-term damage to proteins by glycation and oxidation reactions. PMID:1904067

The nuclear import factor importin α4 can protect against oxidative stress.

PubMed

Young, Julia C; Ly-Huynh, Jennifer D; Lescesen, Helen; Miyamoto, Yoichi; Browne, Cate; Yoneda, Yoshihiro; Koopman, Peter; Loveland, Kate L; Jans, David A

2013-10-01

The importin (IMP) superfamily of nuclear transport proteins is essential to key developmental pathways, including in the murine testis where expression of the 6 distinct IMPα proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMPα4 is unique in showing a striking nuclear localization, a property we previously found to be linked to maintenance of pluripotency in embryonic stem cells and to the cellular stress response in cultured cells. Here we examine the role of IMPα4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMPα4-EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMPα4 expression in spermatids. IMPα4 overexpression did not affect overall fertility, testis morphology/weight or spermatogenic progression under normal conditions, but conferred significantly (>30%) increased resistance to oxidative stress specifically in the spermatid subpopulation expressing the transgene. Consistent with a cell-specific role for IMPα4 in protecting against oxidative stress, haploid germ cells from IMPα4 null mice were significantly (c. 30%) less resistant to oxidative stress than wild type controls. These results from two unique and complementary mouse models demonstrate a novel protective role for IMPα4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity. Copyright © 2013 Elsevier B.V. All rights reserved.

A role for tyrosinase-related protein 1 in 4-tert-butylphenol-induced toxicity in melanocytes: Implications for vitiligo.

PubMed

Manga, Prashiela; Sheyn, David; Yang, Fan; Sarangarajan, Rangaprasad; Boissy, Raymond E

2006-11-01

Vitiligo presents with depigmented cutaneous lesions following localized melanocyte death. Multiple factors contribute to cell death, including genetically determined susceptibility to trauma, and environmental factors, such as exposure to 4-tert-butylphenol (4-TBP). We demonstrate that 4-TBP induces oxidative stress that is more readily overcome by melanocytes from normally pigmented individuals than from two individuals with vitiligo. The antioxidant catalase selectively and significantly reduced death of melanocytes derived from two individuals with vitiligo, indicating a role for oxidative stress in vitiligo pathogenesis. In normal melanocytes, oxidative stress results in reduced expression of microphthalmia-associated transcription factor (MITF). Melanocyte-stimulating hormone-induced expression of MITF protein caused increased sensitivity to 4-TBP, whereas sensitivity of melanomas correlated with MITF expression. MITF stimulates melanin synthesis by up-regulating expression of melanogenic enzymes such as tyrosinase-related protein-1 (Tyrp1). Although melanin content per se did not affect sensitivity to 4-TBP, expression of Tyrp1 significantly increased sensitivity. Melanocytes and melanomas that express functional Tyrp1 were significantly more sensitive to 4-TBP than Tyrp1-null cells. Thus, normal melanocytes respond to 4-TBP by reducing expression of MITF and Tyrp1. We hypothesize that melanocytes in vitiligo demonstrate reduced ability to withstand oxidative stress due, partly, to a disruption in MITF regulation of Tyrp1.

Milk protein composition and stability changes affected by iron in water sources.

PubMed

Wang, Aili; Duncan, Susan E; Knowlton, Katharine F; Ray, William K; Dietrich, Andrea M

2016-06-01

Water makes up more than 80% of the total weight of milk. However, the influence of water chemistry on the milk proteome has not been extensively studied. The objective was to evaluate interaction of water-sourced iron (low, medium, and high levels) on milk proteome and implications on milk oxidative state and mineral content. Protein composition, oxidative stability, and mineral composition of milk were investigated under conditions of iron ingestion through bovine drinking water (infused) as well as direct iron addition to commercial milk in 2 studies. Four ruminally cannulated cows each received aqueous infusions (based on water consumption of 100L) of 0, 2, 5, and 12.5mg/L Fe(2+) as ferrous lactate, resulting in doses of 0, 200, 500 or 1,250mg of Fe/d, in a 4×4Latin square design for a 14-d period. For comparison, ferrous sulfate solution was directly added into commercial retail milk at the same concentrations: control (0mg of Fe/L), low (2mg of Fe/L), medium (5mg of Fe/L), and high (12.5mg of Fe/L). Two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF/TOF) high-resolution tandem mass spectrometry analysis was applied to characterize milk protein composition. Oxidative stability of milk was evaluated by the thiobarbituric acid reactive substances (TBARS) assay for malondialdehyde, and mineral content was measured by inductively coupled plasma mass spectrometry. For milk from both abomasal infusion of ferrous lactate and direct addition of ferrous sulfate, an iron concentration as low as 2mg of Fe/L was able to cause oxidative stress in dairy cattle and infused milk, respectively. Abomasal infusion affected both caseins and whey proteins in the milk, whereas direct addition mainly influenced caseins. Although abomasal iron infusion did not significantly affect oxidation state and mineral balance (except iron), it induced oxidized off-flavor and partial degradation of whey proteins. Direct iron addition to milk led to lipid oxidation during storage at 4°C. Oxidation level was positively associated with the concentration of added iron. Minerals (Mg, P, Na, K, Ca, Zn) in milk were not affected by the added iron in milk. This study indicated that a small amount of iron contamination in bovine drinking water at the farm or incidental iron addition from potable water sources causes oxidation, affects milk protein composition and stability, and affects final milk quality. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Neutrophil-generated oxidative stress and protein damage in Staphylococcus aureus

PubMed Central

Beavers, William N.; Skaar, Eric P.

2016-01-01

Staphylococcus aureus is a ubiquitous, versatile and dangerous pathogen. It colonizes over 30% of the human population, and is one of the leading causes of death by an infectious agent. During S. aureus colonization and invasion, leukocytes are recruited to the site of infection. To combat S. aureus, leukocytes generate an arsenal of reactive species including superoxide, hydrogen peroxide, nitric oxide and hypohalous acids that modify and inactivate cellular macromolecules, resulting in growth defects or death. When S. aureus colonization cannot be cleared by the immune system, antibiotic treatment is necessary and can be effective. Yet, this organism quickly gains resistance to each new antibiotic it encounters. Therefore, it is in the interest of human health to acquire a deeper understanding of how S. aureus evades killing by the immune system. Advances in this field will have implications for the design of future S. aureus treatments that complement and assist the host immune response. In that regard, this review focuses on how S. aureus avoids host-generated oxidative stress, and discusses the mechanisms used by S. aureus to survive oxidative damage including antioxidants, direct repair of damaged proteins, sensing oxidant stress and transcriptional changes. This review will elucidate areas for studies to identify and validate future antimicrobial targets. PMID:27354296

Oxidative stress is a consequence, not a cause, of aluminum toxicity in the forage legume Lotus corniculatus.

PubMed

Navascués, Joaquín; Pérez-Rontomé, Carmen; Sánchez, Diego H; Staudinger, Christiana; Wienkoop, Stefanie; Rellán-Álvarez, Rubén; Becana, Manuel

2012-02-01

• Aluminum (Al) toxicity is a major limiting factor of crop production on acid soils, but the implication of oxidative stress in this process is controversial. A multidisciplinary approach was used here to address this question in the forage legume Lotus corniculatus. • Plants were treated with low Al concentrations in hydroponic culture, and physiological and biochemical parameters, together with semiquantitative metabolic and proteomic profiles, were determined. • The exposure of plants to 10 μM Al inhibited root and leaf growth, but had no effect on the production of reactive oxygen species or lipid peroxides. By contrast, exposure to 20 μM Al elicited the production of superoxide radicals, peroxide and malondialdehyde. In response to Al, there was a progressive replacement of the superoxide dismutase isoforms in the cytosol, a loss of ascorbate and consistent changes in amino acids, sugars and associated enzymes. • We conclude that oxidative stress is not a causative factor of Al toxicity. The increased contents in roots of two powerful Al chelators, malic and 2-isopropylmalic acids, together with the induction of an Al-activated malate transporter gene, strongly suggest that both organic acids are implicated in Al detoxification. The effects of Al on key proteins involved in cytoskeleton dynamics, protein turnover, transport, methylation reactions, redox control and stress responses underscore a metabolic dysfunction, which affects multiple cellular compartments, particularly in plants exposed to 20 μM Al. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

Myeloperoxidase-mediated Methionine Oxidation Promotes an Amyloidogenic Outcome for Apolipoprotein A-I*

PubMed Central

Chan, Gary K. L.; Witkowski, Andrzej; Gantz, Donald L.; Zhang, Tianqi O.; Zanni, Martin T.; Jayaraman, Shobini; Cavigiolio, Giorgio

2015-01-01

High plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, whereas dysfunctional apoA-I is a cause of atherosclerosis. In the atherosclerotic plaques, amyloid deposition increases with aging. Notably, apoA-I is the main component of these amyloids. Recent studies identified high levels of oxidized lipid-free apoA-I in atherosclerotic plaques. Likely, myeloperoxidase (MPO) secreted by activated macrophages in atherosclerotic lesions is the promoter of such apoA-I oxidation. We hypothesized that apoA-I oxidation by MPO levels similar to those present in the artery walls in atherosclerosis can promote apoA-I structural changes and amyloid fibril formation. ApoA-I was exposed to exhaustive chemical (H2O2) oxidation or physiological levels of enzymatic (MPO) oxidation and incubated at 37 °C and pH 6.0 to induce fibril formation. Both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after a few hours of incubation. The amyloid fibrils were composed of full-length apoA-I with differential oxidation of the three methionines. Met to Leu apoA-I variants were used to establish the predominant role of oxidation of Met-86 and Met-148 in the fibril formation process. Importantly, a small amount of preformed apoA-I fibrils was able to seed amyloid formation in oxidized apoA-I at pH 7.0. In contrast to hereditary amyloidosis, wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus, MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid deposition in the atherosclerotic plaques in vivo. PMID:25759391

Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells.

PubMed

Voisin, Grégory; Bouvet, Guillaume F; Legendre, Pierre; Dagenais, André; Massé, Chantal; Berthiaume, Yves

2014-09-01

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients. Copyright © 2014 the American Physiological Society.

Proteome-Level Analysis of Metabolism- and Stress-Related Proteins during Seed Dormancy and Germination in Gnetum parvifolium.

PubMed

Chang, Ermei; Deng, Nan; Zhang, Jin; Liu, Jianfeng; Chen, Lanzhen; Zhao, Xiulian; Abbas, M; Jiang, Zeping; Shi, Shengqing

2018-03-21

Gnetum parvifolium is a rich source of materials for traditional medicines, food, and oil, but little is known about the mechanism underlying its seed dormancy and germination. In this study, we analyzed the proteome-level changes in its seeds during germination using isobaric tags for relative and absolute quantitation. In total, 1,040 differentially expressed proteins were identified, and cluster analysis revealed the distinct time points during which signal transduction and oxidation-reduction activity changed. Gene Ontology analysis showed that "carbohydrate metabolic process" and "response to oxidative stress" were the main enriched terms. Proteins associated with starch degradation and antioxidant enzymes were important for dormancy-release, while proteins associated with energy metabolism and protein synthesis were up-regulated during germination. Moreover, protein-interaction networks were mainly associated with heat-shock proteins. Furthermore, in accord with changes in the energy metabolism- and antioxidant-related proteins, indole-3-acetic acid, Peroxidase, and soluble sugar content increased, and the starch content decreased in almost all six stages of dormancy and germination analyzed (S1-S6). The activity of superoxide dismutase, abscisic acid, and malondialdehyde content increased in the dormancy stages (S1-S3) and then decreased in the germination stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy and germination at the proteome and physiological levels, with implications for improving seed propagation.

Oxidative stress induced by potassium bromate exposure results in altered tight junction protein expression in renal proximal tubule cells.

PubMed

Limonciel, Alice; Wilmes, Anja; Aschauer, Lydia; Radford, Robert; Bloch, Katarzyna M; McMorrow, Tara; Pfaller, Walter; van Delft, Joost H; Slattery, Craig; Ryan, Michael P; Lock, Edward A; Jennings, Paul

2012-11-01

Potassium bromate (KBrO(3)) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO(3) in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO(3) for 6, 24 and 72 h. Pathway analysis identified "glutathione metabolism", "Nrf2-mediated oxidative stress" and "tight junction (TJ) signalling" as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO(3) exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N-Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.

PML nuclear bodies: from architecture to function.

PubMed

Lallemand-Breitenbach, Valérie; de Thé, Hugues

2018-06-01

PML nuclear bodies are nucleated by the PML protein, which polymerizes into spherical shells where it concentrates many unrelated partner proteins. Emerging data has connected PML bodies to post-translational control, notably conjugation by SUMOs. High concentrations of SUMO-bound proteins were proposed to condense into liquid-like droplets and such phase transition may occur within NBs. Many stress pathways modulate NB formation and recent findings have directly implicated PML in oxidative stress response in vivo. PML may also undergo SUMO-dependent ubiquitination/degradation. We highlight recent advances linking PML to partner degradation and other adaptative post-translational modifications in the context of chromatin remodeling, telomere biology, senescence or viral infections. Copyright © 2018. Published by Elsevier Ltd.

KDM5 Interacts with Foxo to Modulate Cellular Levels of Oxidative Stress

PubMed Central

Liu, Xingyin; Greer, Christina; Secombe, Julie

2014-01-01

Increased cellular levels of oxidative stress are implicated in a large number of human diseases. Here we describe the transcription co-factor KDM5 (also known as Lid) as a new critical regulator of cellular redox state. Moreover, this occurs through a novel KDM5 activity whereby it alters the ability of the transcription factor Foxo to bind to DNA. Our microarray analyses of kdm5 mutants revealed a striking enrichment for genes required to regulate cellular levels of oxidative stress. Consistent with this, loss of kdm5 results in increased sensitivity to treatment with oxidizers, elevated levels of oxidized proteins, and increased mutation load. KDM5 activates oxidative stress resistance genes by interacting with Foxo to facilitate its recruitment to KDM5-Foxo co-regulated genes. Significantly, this occurs independently of KDM5's well-characterized demethylase activity. Instead, KDM5 interacts with the lysine deacetylase HDAC4 to promote Foxo deacetylation, which affects Foxo DNA binding. PMID:25329053

Differentially expressed proteins in nitric oxide-stimulated NIH/3T3 fibroblasts: implications for inhibiting cancer development.

PubMed

Shim, Dong Hwi; Lim, Joo Weon; Kim, Hyeyoung

2015-03-01

Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. The cells were treated with 300 μM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (α1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (ε subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (ε subunit) are unknown in relation to carcinogenesis. Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in human hepatoma cells.

PubMed

Ma, Wenbo; Li, Caiyue; Yin, Shikui; Liu, Jingxin; Gao, Chao; Lin, Zuoxian; Huang, Rongqi; Huang, Jufang; Li, Zhiyuan

2015-12-01

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H2O2-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H2O2-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H2O2-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H2O2-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H2O2-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Paraoxonase: The Universal Factor of Antioxidant Defense in Human Body.

PubMed

Borovkova, E I; Antipova, N V; Komeenko, T V; Shakhparonov, M I; Borovkov, I M

The paraoxonase (PON) gene family includes three members: PON1, PON2, and PON3 aligned in tandem on chromosome 7 in humans. All PON proteins share considerable structural homology and have the capacity to protect cells from oxidative stress; therefore, they have been implicated in the pathogenesis of several inflammatory diseases, particularly atherosclerosis. Increased production of reactive oxygen species as a result of decreased activities of mitochondrial electron transport chain complexes plays a role in the development of many inflammatory diseases, including atherosclerosis. PON1 and PON3 proteins can be detected in plasma and reside in the high-density lipoprotein fraction and protect against oxidative stress by hydrolyzing certain oxidized lipids in lipoproteins, macrophages, and atherosclerotic lesions. Paraoxonase 2 (PON2) possesses antiatherogenic properties and is associated with lower ROS levels. PON2 is involved in the antioxidative and anti-inflammatory response in intestinal epithelial cells. In contrast to PON1 and PON3, PON2 is cell-associated and is not found in plasma. It is widely expressed in a variety of tissues, including the kidney, and protects against cellular oxidative stress. Overexpression of PON2 reduces oxidative status, prevents apoptosis in vascular endothelial cells, and inhibits cell-mediated low density lipoprotein oxidation. PON2 also inhibits the development of atherosclerosis, via mechanisms involving the reduction of oxidative stress. In this review we explore the physiological roles of PON in disease development and modulation of PONs by infective (bacterial, viral) agents.

Mitochondrial Translation and Beyond: Processes Implicated in Combined Oxidative Phosphorylation Deficiencies

PubMed Central

Smits, Paulien; Smeitink, Jan; van den Heuvel, Lambert

2010-01-01

Mitochondrial disorders are a heterogeneous group of often multisystemic and early fatal diseases, which are amongst the most common inherited human diseases. These disorders are caused by defects in the oxidative phosphorylation (OXPHOS) system, which comprises five multisubunit enzyme complexes encoded by both the nuclear and the mitochondrial genomes. Due to the multitude of proteins and intricacy of the processes required for a properly functioning OXPHOS system, identifying the genetic defect that underlies an OXPHOS deficiency is not an easy task, especially in the case of combined OXPHOS defects. In the present communication we give an extensive overview of the proteins and processes (in)directly involved in mitochondrial translation and the biogenesis of the OXPHOS system and their roles in combined OXPHOS deficiencies. This knowledge is important for further research into the genetic causes, with the ultimate goal to effectively prevent and cure these complex and often devastating disorders. PMID:20396601

Diet Bioactive Compounds: Implications for Oxidative Stress and Inflammation in the Vascular System.

PubMed

Gabriele, Morena; Pucci, Laura

2017-11-16

Increasing evidence has demonstrated that dietary products and their active components are independently or jointly responsible for the apparent reduction of the cardiovascular diseases (CVDs) risk. Nowadays, there is a growing attention in the use of nutraceuticals as a new approach for the prevention and management of many diseases, as well as for controlling rising of chronic illnesses with minimal side effects. Food-derived peptides, as well as peptide-rich protein hydrolysates, represent new and valuable tools for the prevention of metabolic and cardiovascular diseases, acting as modulators of oxidative stress, inflammation, and overactivity of the renin-angiotensin system (RAS). This review summarizes the recently published data on antioxidant, anti-inflammatory, and vascular protective properties of nutraceuticals, notably on the effects of food-derived bioactive peptides and protein hydrolysates, paying particular attention to those derived from fermented foods. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

PubMed Central

McLelland, Gian-Luca; Soubannier, Vincent; Chen, Carol X; McBride, Heidi M; Fon, Edward A

2014-01-01

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondria-derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin- and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD. PMID:24446486

Mechanism of nitrite oxidation by eosinophil peroxidase: implications for oxidant production and nitration by eosinophils

PubMed Central

van Dalen, Christine J.; Winterbourn, Christine C.; Kettle, Anthony J.

2005-01-01

Eosinophil peroxidase is a haem enzyme of eosinophils that is implicated in oxidative tissue injury in asthma. It uses hydrogen peroxide to oxidize thiocyanate and bromide to their respective hypohalous acids. Nitrite is also a substrate for eosinophil peroxidase. We have investigated the mechanisms by which the enzyme oxidizes nitrite. Nitrite was very effective at inhibiting hypothiocyanous acid (‘cyanosulphenic acid’) and hypobromous acid production. Spectral studies showed that nitrite reduced the enzyme to its compound II form, which is a redox intermediate containing FeIV in the haem active site. Compound II does not oxidize thiocyanate or bromide. These results demonstrate that nitrite is readily oxidized by compound I, which contains FeV at the active site. However, it reacts more slowly with compound II. The observed rate constant for reduction of compound II by nitrite was determined to be 5.6×103 M−1·s−1. Eosinophils were at least 4-fold more effective at promoting nitration of a heptapeptide than neutrophils. This result is explained by our finding that nitrite reacts 10-fold faster with compound II of eosinophil peroxidase than with the analogous redox intermediate of myeloperoxidase. Nitration by eosinophils was increased 3-fold by superoxide dismutase, which indicates that superoxide interferes with nitration. We propose that at sites of eosinophilic inflammation, low concentrations of nitrite will retard oxidant production by eosinophil peroxidase, whereas at higher concentrations nitrogen dioxide will be a major oxidant formed by these cells. The efficiency of protein nitration will be decreased by the diffusion-controlled reaction of superoxide with nitrogen dioxide. PMID:16336215

Different effect of hydrogelation on anti-fouling and circulation properties of dextran–iron oxide nanoparticles

PubMed Central

Karmali, Priya Prakash; Chao, Ying; Park, Ji-Ho (Joe); Sailor, Michael J.; Ruoslahti, Erkki; Esener, Sadik C.; Simberg, Dmitri

2012-01-01

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this “stealth” effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by crosslinking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages in vivo. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that: (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles’ long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles. PMID:22243419

Effects of an Exercise Intervention on Cancer-Related Fatigue and Its Relationship to Markers of Oxidative Stress.

PubMed

Repka, Chris P; Hayward, Reid

2018-06-01

Although the underlying mechanisms of cancer-related fatigue (CRF) are not fully characterized, treatment-associated oxidative stress may play a role. The purpose of this study was to determine the effect of an exercise intervention on the relationship between CRF and oxidative stress. Upon cessation of radiation or chemotherapy, 8 cancer patients participated in a 10-week exercise intervention (EX), while 7 continued standard care (CON). Blood draws and fatigue questionnaires were administered to cancer patients before and after the intervention as well as to 7 age-matched individuals with no cancer history. Changes in plasma 8-hydroxy-deoxyguanosine (8-OHdG), protein carbonyls, antioxidant capacity, and fatigue were compared between groups. Correlations between CRF and oxidative stress were evaluated. Mean total fatigue scores decreased significantly (5.0 ± 2.2 to 2.6 ± 1.5, P < .05) in EX, but not in CON. Antioxidant capacity significantly increased (+41%; P < .05) and protein carbonyls significantly decreased (-36%; P < .05) in EX, but not in CON. Increases in antioxidant capacity were significantly correlated with reductions in affective ( r = -.49), sensory ( r = -.47), and cognitive fatigue ( r = -.58). Changes in total ( r = .46) and affective ( r = .47) fatigue exhibited significant correlations with changes in 8-OHdG over time, while behavioral ( r = .46) and sensory ( r = .47) fatigue changes were significantly correlated with protein carbonyls. Oxidative stress may be implicated in CRF, while improved antioxidant capacity following an exercise intervention may play a role in mitigating CRF in cancer survivors.

Effects of mutations in the human uncoupling protein 3 gene on the respiratory quotient and fat oxidation in severe obesity and type 2 diabetes.

PubMed Central

Argyropoulos, G; Brown, A M; Willi, S M; Zhu, J; He, Y; Reitman, M; Gevao, S M; Spruill, I; Garvey, W T

1998-01-01

Human uncoupling protein 3 (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative ATP phosphorylation. With the capacity to participate in thermogenesis and energy balance, UCP3 is an important obesity candidate gene. A missense polymorphism in exon 3 (V102I) was identified in an obese and diabetic proband. A mutation introducing a stop codon in exon 4 (R143X) and a terminal polymorphism in the splice donor junction of exon 6 were also identified in a compound heterozygote that was morbidly obese and diabetic. Allele frequencies of the exon 3 and exon 6 splice junction polymorphisms were determined and found to be similar in Gullah-speaking African Americans and the Mende tribe of Sierra Leone, but absent in Caucasians. Moreover, in exon 6-splice donor heterozygotes, basal fat oxidation rates were reduced by 50%, and the respiratory quotient was markedly increased compared with wild-type individuals, implicating a role for UCP3 in metabolic fuel partitioning. PMID:9769326

Sirtuins: molecular traffic lights in the crossroad of oxidative stress, chromatin remodeling, and transcription.

PubMed

Rajendran, Ramkumar; Garva, Richa; Krstic-Demonacos, Marija; Demonacos, Constantinos

2011-01-01

Transcription is regulated by acetylation/deacetylation reactions of histone and nonhistone proteins mediated by enzymes called KATs and HDACs, respectively. As a major mechanism of transcriptional regulation, protein acetylation is a key controller of physiological processes such as cell cycle, DNA damage response, metabolism, apoptosis, and autophagy. The deacetylase activity of class III histone deacetylases or sirtuins depends on the presence of NAD(+) (nicotinamide adenine dinucleotide), and therefore, their function is closely linked to cellular energy consumption. This activity of sirtuins connects the modulation of chromatin dynamics and transcriptional regulation under oxidative stress to cellular lifespan, glucose homeostasis, inflammation, and multiple aging-related diseases including cancer. Here we provide an overview of the recent developments in relation to the diverse biological activities associated with sirtuin enzymes and stress responsive transcription factors, DNA damage, and oxidative stress and relate the involvement of sirtuins in the regulation of these processes to oncogenesis. Since the majority of the molecular mechanisms implicated in these pathways have been described for Sirt1, this sirtuin family member is more extensively presented in this paper.

Response to reactive nitrogen intermediates in Mycobacterium tuberculosis: induction of the 16-kilodalton alpha-crystallin homolog by exposure to nitric oxide donors.

PubMed

Garbe, T R; Hibler, N S; Deretic, V

1999-01-01

In contrast to the apparent paucity of Mycobacterium tuberculosis response to reactive oxygen intermediates, this organism has evolved a specific response to nitric oxide challenge. Exposure of M. tuberculosis to NO donors induces the synthesis of a set of polypeptides that have been collectively termed Nox. In this work, the most prominent Nox polypeptide, Nox16, was identified by immunoblotting and by N-terminal sequencing as the alpha-crystallin-related, 16-kDa small heat shock protein, sHsp16. A panel of chemically diverse donors of nitric oxide, with the exception of nitroprusside, induced sHsp16 (Nox16). Nitroprusside, a coordination complex of Fe2+ with a nitrosonium (NO+) ion, induced a 19-kDa polypeptide (Nox19) homologous to the nonheme bacterial ferritins. We conclude that the NO response in M. tuberculosis is dominated by increased synthesis of the alpha-crystallin homolog sHsp16, previously implicated in stationary-phase processes and found in this study to be a major M. tuberculosis protein induced upon exposure to reactive nitrogen intermediates.

Potential Role of Protein Disulfide Isomerase in Metabolic Syndrome-Derived Platelet Hyperactivity

PubMed Central

Gaspar, Renato Simões

2016-01-01

Metabolic Syndrome (MetS) has become a worldwide epidemic, alongside with a high socioeconomic cost, and its diagnostic criteria must include at least three out of the five features: visceral obesity, hypertension, dyslipidemia, insulin resistance, and high fasting glucose levels. MetS shows an increased oxidative stress associated with platelet hyperactivation, an essential component for thrombus formation and ischemic events in MetS patients. Platelet aggregation is governed by the peroxide tone and the activity of Protein Disulfide Isomerase (PDI) at the cell membrane. PDI redox active sites present active cysteine residues that can be susceptible to changes in plasma oxidative state, as observed in MetS. However, there is a lack of knowledge about the relationship between PDI and platelet hyperactivation under MetS and its metabolic features, in spite of PDI being a mediator of important pathways implicated in MetS-induced platelet hyperactivation, such as insulin resistance and nitric oxide dysfunction. Thus, the aim of this review is to analyze data available in the literature as an attempt to support a possible role for PDI in MetS-induced platelet hyperactivation. PMID:28053690

Potential Role of Protein Disulfide Isomerase in Metabolic Syndrome-Derived Platelet Hyperactivity.

PubMed

Gaspar, Renato Simões; Trostchansky, Andrés; Paes, Antonio Marcus de Andrade

2016-01-01

Metabolic Syndrome (MetS) has become a worldwide epidemic, alongside with a high socioeconomic cost, and its diagnostic criteria must include at least three out of the five features: visceral obesity, hypertension, dyslipidemia, insulin resistance, and high fasting glucose levels. MetS shows an increased oxidative stress associated with platelet hyperactivation, an essential component for thrombus formation and ischemic events in MetS patients. Platelet aggregation is governed by the peroxide tone and the activity of Protein Disulfide Isomerase (PDI) at the cell membrane. PDI redox active sites present active cysteine residues that can be susceptible to changes in plasma oxidative state, as observed in MetS. However, there is a lack of knowledge about the relationship between PDI and platelet hyperactivation under MetS and its metabolic features, in spite of PDI being a mediator of important pathways implicated in MetS-induced platelet hyperactivation, such as insulin resistance and nitric oxide dysfunction. Thus, the aim of this review is to analyze data available in the literature as an attempt to support a possible role for PDI in MetS-induced platelet hyperactivation.

Cloning of nitric oxide associated 1 (NOA1) transcript from oil palm (Elaeis guineensis) and its expression during Ganoderma infection.

PubMed

Kwan, Yee-Min; Meon, Sariah; Ho, Chai-Ling; Wong, Mui-Yun

2015-02-01

Nitric oxide associated 1 (NOA1) protein is implicated in plant disease resistance and nitric oxide (NO) biosynthesis. A full-length cDNA encoding of NOA1 protein from oil palm (Elaeis guineensis) was isolated and designated as EgNOA1. Sequence analysis suggested that EgNOA1 was a circular permutated GTPase with high similarity to the bacterial YqeH protein of the YawG/YlqF family. The gene expression of EgNOA1 and NO production in oil palm root tissues treated with Ganoderma boninense, the causal agent of basal stem rot (BSR) disease were profiled to investigate the involvement of EgNOA1 during fungal infection and association with NO biosynthesis. Real-time PCR (qPCR) analysis revealed that the transcript abundance of EgNOA1 in root tissues was increased by G. boninense treatment. NO burst in Ganoderma-treated root tissue was detected using Griess reagent, in advance of the up-regulation of the EgNOA1 transcript. This indicates that NO production was independent of EgNOA1. However, the induced expression of EgNOA1 in Ganoderma-treated root tissues implies that it might be involved in plant defense responses against pathogen infection. Copyright © 2014 Elsevier GmbH. All rights reserved.

Studying Prokaryotic Communities in Iron Depositing Hot Springs (IDHS): Implication for Early Mars Habitability

NASA Technical Reports Server (NTRS)

Sarkisova, S. A.; Tringe, S. G.; Thomas-Keprta, K. L.; Allen, C. c.; Garrison, D. H.; McKay, David S.; Brown, I. I.

2010-01-01

We speculate that both external and intracellular iron precipitate in iron-tolerant CB might be involved in oxidative stress suppression shown by [9]. Significant differences are apparent between a set of proteins involved in the maintenance of Fe homeostasis and oxidative stress protection in iron-tolerant and fresh-water and marine CB. Correspondingly, these properties may help to make iron-tolerant CB as dominant organisms in IDHS and probably on early Earth and Mars. Further comparative analyses of hot springs metagenomes and the genomes of iron-tolerant microbes versus fresh-water/marine ones may point out to different habitable zones on early Mars.

Rational engineering of Geobacter sulfurreducens electron transfer components: A foundation for building improved Geobacter-based bioelectrochemical technologies

DOE PAGES

Dantas, Joana M.; Morgado, Leonor; Aklujkar, Muktak; ...

2015-07-30

Multiheme cytochromes have been implicated in Geobacter sulfurreducens extracellular electron transfer (EET). These proteins are potential targets to improve EET and enhance bioremediation and electrical current production by G. sulfurreducens. However, the functional characterization of multiheme cytochromes is particularly complex due to the co-existence of several microstates in solution, connecting the fully reduced and fully oxidized states. Throughout the last decade, new strategies have been developed to characterize multiheme redox proteins functionally and structurally. These strategies were used to reveal the functional mechanism of G. sulfurreducens multiheme cytochromes and also to identify key residues in these proteins for EET. Inmore » previous studies, we set the foundations for enhancement of the EET abilities of G. sulfurreducens by characterizing a family of five triheme cytochromes (PpcA-E). These periplasmic cytochromes are implicated in electron transfer between the oxidative reactions of metabolism in the cytoplasm and the reduction of extracellular terminal electron acceptors at the cell's outer surface. The results obtained suggested that PpcA can couple e -/H + transfer, a property that might contribute to the proton electrochemical gradient across the cytoplasmic membrane for metabolic energy production. The structural and functional properties of PpcA were characterized in detail and used for rational design of a family of 23 single site PpcA mutants. In this review, we summarize the functional characterization of the native and mutant proteins. Mutants that retain the mechanistic features of PpcA and adopt preferential e -/H + transfer pathways at lower reduction potential values compared to the wild-type protein were selected for in vivo studies as the best candidates to increase the electron transfer rate of G. sulfurreducens. For the first time G. sulfurreducens strains have been manipulated by the introduction of mutant forms of essential proteins with the aim to develop and improve bioelectrochemical technologies.« less

Nutritional value and digestion rate of rhea meat proteins in association with storage and cooking processes.

PubMed

Filgueras, Renata S; Gatellier, Philippe; Ferreira, Claude; Zambiazi, Rui C; Santé-Lhoutellier, Véronique

2011-09-01

The nutritional value of proteins was investigated after the storage and cooking of rhea M. Gastrocnemius pars interna. Oxidation of basic and aromatic amino acids, surface hydrophobicity and aggregation state of proteins, were determined in raw and cooked meat. In addition, myofibrillar proteins were exposed in vitro to proteases of the digestive tract. Cooking markedly affected the protein surface hydrophobicity. The BBP bound content was three times greater in cooked than in fresh rhea meat. A small increment in tryptophan content after cooking was observed. Storage influenced Schiff bases formation indicating the presence of protein-aldehyde adducts after cooking. High content of Schiff bases was found after cooking of samples stored for 5 days, demonstrating a probable implication of free amino groups, most likely from lysine. Cooking decreased the myofibrillar protein susceptibility to pepsin activity. After cooking, the proteolysis rate by pancreatic enzymes increased. Our findings support the importance of protein aggregation in the nutritional value of meat proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection

PubMed Central

Ganini, Douglas; Canistro, Donatella; Jang, JinJie; Stadler, Krisztian; Mason, Ronald P.; Kadiiska, Maria B.

2012-01-01

Ceruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe2+ oxidation in plasma of mammals. Besides its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) and related probes in Chelex-treated phosphate buffer and rat serum. The reaction is suppressed by the metal chelators DTPA, EDTA and Desferal, while heparin and bathocuproine have no effect. Catalase or SOD additions do not interfere with the CPH-oxidation yield, demonstrating that free radicals are not involved in the CPH oxidation mediated by ceruloplasmin. Plasma samples immunodepleted of ceruloplasmin have lower levels of CPH oxidation, which confirms the role of ceruloplasmin (ferroxidase) as a biological oxidizing agent of cyclic hydroxylamines. In conclusion, we show that the ferroxidase activity of ceruloplasmin is a possible biological source of artifacts in the cyclic hydroxylamine-oxidation assay used for ROS detection and quantification. PMID:22824865

The Moraxella catarrhalis nitric oxide reductase is essential for nitric oxide detoxification.

PubMed

Wang, Wei; Kinkel, Traci; Martens-Habbena, Willm; Stahl, David A; Fang, Ferric C; Hansen, Eric J

2011-06-01

Moraxella catarrhalis is a Gram-negative obligate aerobe that is an important cause of human respiratory tract infections. The M. catarrhalis genome encodes a predicted truncated denitrification pathway that reduces nitrate to nitrous oxide. We have previously shown that expression of both the M. catarrhalis aniA (encoding a nitrite reductase) and norB (encoding a putative nitric oxide reductase) genes is repressed by the transcriptional regulator NsrR under aerobic conditions and that M. catarrhalis O35E nsrR mutants are unable to grow in the presence of low concentrations of nitrite (W. Wang, et al., J. Bacteriol. 190:7762-7772, 2008). In this study, we constructed an M. catarrhalis norB mutant and showed that planktonic growth of this mutant is inhibited by low levels of nitrite, whether or not an nsrR mutation is present. To determine the importance of NorB in this truncated denitrification pathway, we analyzed the metabolism of nitrogen oxides by norB, aniA norB, and nsrR norB mutants. We found that norB mutants are unable to reduce nitric oxide and produce little or no nitrous oxide from nitrite. Furthermore, nitric oxide produced from nitrite by the AniA protein is bactericidal for a Moraxella catarrhalis O35E norB mutant but not for wild-type O35E bacteria under aerobic growth conditions in vitro, suggesting that nitric oxide catabolism in M. catarrhalis is accomplished primarily by the norB gene product. Measurement of bacterial protein S-nitrosylation directly implicates nitrosative stress resulting from AniA-dependent nitric oxide formation as a cause of the growth inhibition of norB and nsrR mutants by nitrite.

Terminal oxidase diversity and function in "Metallosphaera yellowstonensis": gene expression and protein modeling suggest mechanisms of Fe(II) oxidation in the sulfolobales.

PubMed

Kozubal, M A; Dlakic, M; Macur, R E; Inskeep, W P

2011-03-01

"Metallosphaera yellowstonensis" is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowstonensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEFGHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b(558/566), several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that the fox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance of fox genes (e.g., foxA and foxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores.

Effects of rutin on the redox reactions of hemoglobin.

PubMed

Lu, Naihao; Ding, Yun; Yang, Zhen; Gao, Pingzhang

2016-08-01

Flavonoids are widely used to attenuate oxidative damage in vitro and in vivo. In this study, we investigated the influence of rutin (quercetin-3-rhamnosylglucoside) on hemoglobin (Hb)- dependent redox reactions, i.e. oxidative stability of Hb and its cytotoxic ferryl intermediate. It was found that rutin induced generation of H2O2, which in turn oxidized Hb rapidly. Meanwhile, rutin exhibited anti-oxidant effect by effectively reducing ferryl intermediate back to ferric Hb at physiological pH. In comparison with quercetin, rutin had stronger capability on reducing ferryl species while lesser pro-oxidant effect on H2O2 generation, thus it exhibited more protective effect on H2O2-induced Hb oxidation. Circular dichroism spectrum showed no significant change in the secondary structure of Hb after flavonoid addition, while molecular docking revealed different binding modes of quercetin and rutin with Hb. These results might provide new insights into the potential nutritional and physiological implications of rutin and quercetin with redox active heme proteins regarding their ani- and pro-oxidant effects. Copyright © 2016 Elsevier B.V. All rights reserved.

Suppressors of cytokine-signaling proteins induce insulin resistance in the retina and promote survival of retinal cells.

PubMed

Liu, Xuebin; Mameza, Marie G; Lee, Yun Sang; Eseonu, Chikezie I; Yu, Cheng-Rong; Kang Derwent, Jennifer J; Egwuagu, Charles E

2008-06-01

Suppressors of cytokine signaling (SOCS) are implicated in the etiology of diabetes, obesity, and metabolic syndrome. Here, we show that some SOCS members are induced, while others are constitutively expressed, in retina and examine whether persistent elevation of SOCS levels in retina by chronic inflammation or cellular stress predisposes to developing insulin resistance in retina, a condition implicated in diabetic retinopathy. SOCS-mediated insulin resistance and neuroprotection in retina were investigated in 1) an experimental uveitis model, 2) SOCS1 transgenic rats, 3) insulin-deficient diabetic rats, 4) retinal cells depleted of SOCS6 or overexpressing SOCS1/SOCS3, and 5) oxidative stress and light-induced retinal degeneration models. We show that constitutive expression of SOCS6 protein in retinal neurons may improve glucose metabolism, while elevated SOCS1/SOCS3 expression during uveitis induces insulin resistance in neuroretina. SOCS-mediated insulin resistance, as indicated by its inhibition of basally active phosphoinositide 3-kinase/AKT signaling in retina, is validated in retina-specific SOCS1 transgenic rats and retinal cells overexpressing SOCS1/SOCS3. We further show that the SOCS3 level is elevated in retina by oxidative stress, metabolic stress of insulin-deficient diabetes, or light-induced retinal damage and protects ganglion cells from apoptosis, suggesting that upregulation of SOCS3 may be a common physiologic response of neuroretinal cells to cellular stress. Our data suggest two-sided roles of SOCS proteins in retina. Whereas SOCS proteins may improve glucose metabolism, mitigate deleterious effects of inflammation, and promote neuroprotection, persistent SOCS3 expression caused by chronic inflammation or cellular stress can induce insulin resistance and inhibit neurotrophic factors, such as ciliary neurotrophic factor, leukemia inhibitory factor, and insulin, that are essential for retinal cell survival.

Robust Production, Crystallization, Structure Determination, and Analysis of [Fe-S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes.

PubMed

Tsai, Chi-Lin; Tainer, John A

2018-01-01

[Fe-S] clusters are essential cofactors in all domains of life. They play many biological roles due to their unique abilities for electron transfer and conformational control. Yet, producing and analyzing Fe-S proteins can be difficult and even misleading if not done anaerobically. Due to unique redox properties of [Fe-S] clusters and their oxygen sensitivity, they pose multiple challenges and can lose enzymatic activity or cause their component proteins to be structurally disordered due to [Fe-S] cluster oxidation and loss in air. Here we highlight tested protocols and strategies enabling efficient and stable [Fe-S] protein production, purification, crystallization, X-ray diffraction data collection, and structure determination. From multiple high-resolution anaerobic crystal structures, we furthermore analyze exemplary data defining [Fe-S] clusters, substrate entry, and product exit for the functional oxidation states of type II molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) enzymes. Notably, these enzymes perform electron shuttling between quinone pools and specific substrates to catalyze respiratory metabolism. The identified structure-activity relationships for this enzyme class have broad implications germane to perchlorate environments on Earth and Mars extending to an alternative mechanism underlying metabolic origins for the evolution of the oxygen atmosphere. Integrated structural analyses of type II Mo-bisMGD enzymes unveil novel distinctive shared molecular mechanisms for dynamic control of substrate entry and product release gated by hydrophobic residues. Collective findings support a prototypic model for type II Mo-bisMGD enzymes including insights for a fundamental molecular mechanistic understanding of selectivity and regulation by a conformationally gated channel with general implications for [Fe-S] cluster respiratory enzymes. © 2018 Elsevier Inc. All rights reserved.

Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans

PubMed Central

Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

2016-01-01

Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development. PMID:26791749

Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans.

PubMed

Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

2016-01-21

Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development.

Reactive Oxygen Species and the Aging Eye: Specific Role of Metabolically Active Mitochondria in Maintaining Lens Function and in the Initiation of the Oxidation-Induced Maturity Onset Cataract--A Novel Platform of Mitochondria-Targeted Antioxidants With Broad Therapeutic Potential for Redox Regulation and Detoxification of Oxidants in Eye Diseases.

PubMed

Babizhayev, Mark A; Yegorov, Yegor E

2016-01-01

The aging eye appears to be at considerable risk from oxidative stress. A great deal of research indicates that dysfunctional mitochondria are the primary site of reactive oxygen species (ROS). More than 95% of O2 produced during normal metabolism is generated by the electron transport chain in the inner mitochondrial membrane. Mitochondria are also the major target of ROS. Cataract formation, the opacification of the eye lens, is one of the leading causes of human blindness worldwide, accounting for 47.8% of all causes of blindness. Cataracts result from the deposition of aggregated proteins in the eye lens and lens fiber cell plasma membrane damage, which causes clouding of the lens, light scattering, and obstruction of vision. ROS-induced damage in the lens cell may consist of oxidation of proteins, DNA damage, and/or lipid peroxidation, all of which have been implicated in cataractogenesis. This article is an attempt to integrate how mitochondrial ROS are altered in the aging eye along with those protective and repair therapeutic systems believed to regulate ROS levels in ocular tissues and how damage to these systems contributes to age-onset eye disease and cataract formation. Mitochondria-targeted antioxidants might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo. As a result of the combination of weak metal chelating, OH and lipid peroxyl radicals scavenging, reducing activities to liberated fatty acid, and phospholipid hydroperoxides, carnosine and carcinine appear to be physiological antioxidants able to efficiently protect the lipid phase of biologic membranes and aqueous environments and act as the antiapoptotic natural drug compounds The authors developed and patented the new ophthalmic compositions, including N-acetylcarnosine, acting as a prodrug of naturally targeted to mitochondria L-carnosine endowed with pluripotent antioxidant activities combined with mitochondria-targeted rechargeable antioxidant (either MitoVit E, Mito Q, or SkQs) as a potent medicine to treat ocular diseases. Such specificity is explained by the fact that developed compositions might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo and outside mitochondria in the cellular and tissue structures of the lens and eye compartments. Mitochondrial targeting of compounds with universal types of antioxidant activity represents a promising approach for treating a number of ROS-related ocular diseases of the aging eye and can be implicated in the management of cataracts.

Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide

PubMed Central

Fomenko, Dmitri E.; Koc, Ahmet; Agisheva, Natalia; Jacobsen, Michael; Kaya, Alaattin; Malinouski, Mikalai; Rutherford, Julian C.; Siu, Kam-Leung; Jin, Dong-Yan; Winge, Dennis R.; Gladyshev, Vadim N.

2011-01-01

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H2O2 response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H2O2. Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H2O2. The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H2O2 and other cellular proteins plays a secondary role. PMID:21282621

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haque, Rizwanul; Umstead, Todd M.; Ponnuru, Padmavathi

Millions are exposed to ozone levels above recommended limits, impairing lung function, causing epithelial damage and inflammation, and predisposing some individuals to pneumonia, asthma, and other lung conditions. Surfactant protein-A (SP-A) plays a role in host defense, the regulation of inflammation, and repair of tissue damage. We tested the hypothesis that the lungs of SP-A(-/-) (KO) mice are more susceptible to ozone-induced damage. We compared the effects of ozone on KO and wild type (WT) mice on the C57BL/6 genetic background by exposing them to 2 parts/million of ozone for 3 or 6 h and sacrificing them 0, 4, andmore » 24 h later. Lungs were subject to bronchoalveolar lavage (BAL) or used to measure endpoints of oxidative stress and inflammation. Despite more total protein in BAL of KO mice after a 3 h ozone exposure, WT mice had increased oxidation of protein and had oxidized SP-A dimers. In KO mice there was epithelial damage as assessed by increased LDH activity and there was increased phospholipid content. In WT mice there were more BAL PMNs and elevated macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1. Changes in MIP-2 and MCP-1 were observed in both KO and WT, however mRNA levels differed. In KO mice MIP-2 mRNA levels changed little with ozone, but in WT levels they were significantly increased. In summary, several aspects of the inflammatory response differ between WT and KO mice. These in vivo findings appear to implicate SP-A in regulating inflammation and limiting epithelial damage in response to ozone exposure.« less

Cardiac Nitric Oxide Synthases and Na⁺/K⁺-ATPase in the Rat Model of Polycystic Ovary Syndrome Induced by Dihydrotestosterone.

PubMed

Tepavčević, S; Milutinović, D V; Macut, D; Stanišić, J; Nikolić, M; Božić-Antić, I; Rodaljević, S; Bjekić-Macut, J; Matić, G; Korićanac, G

2015-05-01

Nitric oxide synthases (NOSs) and Na(+)/K(+)-ATPase are enzymes essential for regular functioning of the heart. Since both enzymes are under insulin and androgen regulation and since insulin action and androgen level were disturbed in polycystic ovary syndrome (PCOS), we hypothesized that cardiac nitric oxide (NO) production and sodium/potassium transport would be deteriorated in PCOS. To test our hypothesis we introduced animal model of PCOS based on dihydrotestosterone (DHT) treatment of female Wistar rats and analyzed protein expression, phosphorylation or subcellular localization of endothelial NOS (eNOS), inducible NOS (iNOS) and alpha subunits of Na(+)/K(+)-ATPase in the heart. Obtained results indicate that DHT treatment significantly decreased cardiac eNOS protein level and activating phosphorylation at serine 1,177, while inhibitory phosphorylation at threonine 495 was increased. In contrast to expression of eNOS, iNOS protein level in the heart of DHT-treated rats was significantly elevated. Furthermore, cardiac protein level of alpha 1 subunit of the ATPase, as well as its plasma membrane content, were decreased in rats with PCOS. In line with this, alpha 2 subunit protein level in fraction of plasma membranes was also significantly below control level. In conclusion, DHT treatment impaired effectiveness of NOSs and Na(+)/K(+)-ATPase in the female rat heart. Regarding the importance of NO production and sodium/potassium transport in the cardiac contraction and blood flow regulation, it implicates strong consequences of PCOS for heart functioning. © Georg Thieme Verlag KG Stuttgart · New York.

Dysregulated nitric oxide signaling as a candidate mechanism of fragile X syndrome and other neuropsychiatric disorders.

PubMed

Colvin, Steven M; Kwan, Kenneth Y

2014-01-01

A mechanistic understanding of the pathophysiology underpinning psychiatric disorders is essential for the development of targeted molecular therapies. For fragile X syndrome (FXS), recent mechanistic studies have been focused on the metabotropic glutamate receptor (mGluR) signaling pathway. This line of research has led to the discovery of promising candidate drugs currently undergoing various phases of clinical trial, and represents a model of how biological insights can inform therapeutic strategies in neurodevelopmental disorders. Although mGluR signaling is a key mechanism at which targeted treatments can be directed, it is likely to be one of many mechanisms contributing to FXS. A more complete understanding of the molecular and neural underpinnings of the disorder is expected to inform additional therapeutic strategies. Alterations in the assembly of neural circuits in the neocortex have been recently implicated in genetic studies of autism and schizophrenia, and may also contribute to FXS. In this review, we explore dysregulated nitric oxide signaling in the developing neocortex as a novel candidate mechanism of FXS. This possibility stems from our previous work demonstrating that neuronal nitric oxide synthase 1 (NOS1 or nNOS) is regulated by the FXS protein FMRP in the mid-fetal human neocortex. Remarkably, in the mid-late fetal and early postnatal neocortex of human FXS patients, NOS1 expression is severely diminished. Given the role of nitric oxide in diverse neural processes, including synaptic development and plasticity, the loss of NOS1 in FXS may contribute to the etiology of the disorder. Here, we outline the genetic and neurobiological data that implicate neocortical dysfunction in FXS, review the evidence supporting dysregulated nitric oxide signaling in the developing FXS neocortex and its contribution to the disorder, and discuss the implications for targeting nitric oxide signaling in the treatment of FXS and other psychiatric illnesses.

Mitochondrial Molecular Abnormalities Revealed by Proteomic Analysis of Hippocampal Organelles of Mice Triple Transgenic for Alzheimer Disease

PubMed Central

Yu, Haitao; Lin, Xuemei; Wang, Dian; Zhang, Zaijun; Guo, Yi; Ren, Xiaohu; Xu, Benhong; Yuan, Jianhui; Liu, Jianjun; Spencer, Peter S.; Wang, Jian-Zhi; Yang, Xifei

2018-01-01

Mitochondrial dysfunction is implicated in the pathogenesis of Alzheimer’s disease (AD). However, the precise mitochondrial molecular deficits in AD remain poorly understood. Mitochondrial and nuclear proteomic analysis in mature male triple transgenic AD mice (PS1M146V/APPSwe/TauP301L) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF-MS/MS, bio-informatics analysis and immunofluorescent staining were performed in this study. In addition to impaired spatial memory impairment and intracellular accumulation of amyloid 1–42 (Aβ1–42) in the 3xTg-AD mice, a well-accepted mouse model of the human disease, we also found significantly increased DNA oxidative damage in entorhinal cortex, hippocampal CA1, CA3 and dental gyrus (DG), as evidenced by the positive staining of 8-hydroxyguanosine, a biomarker of mild cognitive impairment early in AD. We identified significant differences in 27 hippocampal mitochondrial proteins (11 increased and 16 decreased), and 37 hippocampal nuclear proteins (12 increased and 25 decreased) in 3xTg-AD mice compared with the wild-type (WT) mice. Differentially expressed mitochondrial and nuclear proteins were mainly involved in energy metabolism (>55%), synapses, DNA damage, apoptosis and oxidative stress. Two proteins were differentially expressed in both hippocampal mitochondria and nuclei, namely electron transport chain (ETC)-related protein ATP synthase subunit d (ATP5H) was significantly decreased, and apoptosis-related dynamin-1 (DYN1), a pre-synaptic and mitochondrial division-regulated protein that was significantly increased. In sum, perturbations of hippocampus mitochondrial energy metabolism-related proteins responsible for ATP generation via oxidation phosphorylation (OXPHOS), especially nuclear-encoded OXPHOS proteins, correlated with the amyloid-associated cognitive deficits of this murine AD model. The molecular changes in respiratory chain-related proteins and DYN1 may represent novel biomarkers of AD. PMID:29593495

O-GlcNAcylation and oxidation of proteins: is signalling in the cardiovascular system becoming sweeter?

PubMed Central

Lima, Victor V.; Spitler, Kathryn; Choi, Hyehun; Webb, R. Clinton; Tostes, Rita C.

2012-01-01

O-GlcNAcylation is an unusual form of protein glycosylation, where a single-sugar [GlcNAc (N-acetylglucosamine)] is added (via β-attachment) to the hydroxyl moiety of serine and threonine residues of nuclear and cytoplasmic proteins. A complex and extensive interplay exists between O-GlcNAcylation and phosphorylation. Many phosphorylation sites are also known glycosylation sites, and this reciprocal occupancy may produce different activities or alter the stability in a target protein. The interplay between these two post-translational modifications is not always reciprocal, as some proteins can be concomitantly phosphorylated and O-GlcNAcylated, and the adjacent phosphorylation or O-GlcNAcylation can regulate the addition of either moiety. Increased cardiovascular production of ROS (reactive oxygen species), termed oxidative stress, has been consistently reported in various chronic diseases and in conditions where O-GlcNAcylation has been implicated as a contributing mechanism for the associated organ injury/protection (for example, diabetes, Alzheimer's disease, arterial hypertension, aging and ischaemia). In the present review, we will briefly comment on general aspects of O-GlcNAcylation and provide an overview of what has been reported for this post-translational modification in the cardiovascular system. We will then specifically address whether signalling molecules involved in redox signalling can be modified by O-GlcNAc (O-linked GlcNAc) and will discuss the critical interplay between O-GlcNAcylation and ROS generation. Experimental evidence indicates that the interactions between O-GlcNAcylation and oxidation of proteins are important not only for cell regulation in physiological conditions, but also under pathological states where the interplay may become dysfunctional and thereby exacerbate cellular injury. PMID:22757958

Apigenin attenuates streptozotocin-induced pancreatic β cell damage by its protective effects on cellular antioxidant defense.

PubMed

Wang, Ning; Yi, Wen Jing; Tan, Lu; Zhang, Jia Hui; Xu, Jiamin; Chen, Yi; Qin, Mengting; Yu, Shuang; Guan, Jing; Zhang, Rui

2017-06-01

Pancreatic beta cells are very sensitive to oxidative stress, which is one of the major causes of cell damages in diabetes. Growing interest has focused on the development of effective therapeutics to protect pancreatic cells from oxidative stress and searching for potentially protective antioxidants for treating diabetes. Apigenin, a plant-derived flavonoid, was investigated to determine whether it could protect rat insulinoma cell lines (RINm5F pancreatic beta cells) against streptozotocin (STZ)-induced oxidative damages and the mechanisms implicated. Our results showed that STZ treatment could induce oxidative stress and consequent cytotoxic effects in RINm5F cells. Pretreatment with apigenin effectively decreased the intracellular reactive oxygen species (ROS) production, attenuated cellular DNA damage, diminished lipid peroxidation, relieved protein carbonylation, and restored the cell apoptosis of pancreatic beta cells stressed by STZ. Our further experiments demonstrated that the beneficial effects of apigenin were related to ameliorate the loss of antioxidant enzymes of the STZ-treated cells in the level of gene transcription, protein expression, and enzyme activity. That suggested apigenin was not only a free radical scavenger but also a regulator to antioxidant defenses of pancreatic cells. Taken all together, our findings suggested that apigenin could attenuate the STZ-induced oxidative damages in pancreatic beta cells and might serve as a novel agent for the treatment of diabetes.

Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy

PubMed Central

Qi, Xiaoping; Beli, Eleni; Rao, Haripriya V.; Ding, Jindong; Ip, Colin S.; Gu, Hongmei; Akin, Debra; Dunn, William A.; Bowes Rickman, Catherine; Lewin, Alfred S.; Grant, Maria B.; Boulton, Michael E.

2017-01-01

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress. PMID:28222108

S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death.

PubMed

Kohr, Mark J; Evangelista, Alicia M; Ferlito, Marcella; Steenbergen, Charles; Murphy, Elizabeth

2014-04-01

Oxidative stress and membrane damage following myocardial ischemia/reperfusion injury are important contributors to cardiomyocyte death and the loss of myocardial function. Our previous study identified cysteine 144 (C144) of tripartite motif-containing protein 72 (TRIM72) as a potential site for S-nitrosylation (SNO). TRIM72 is a cardioprotective membrane repair protein that can be both activated and targeted for degradation by different oxidative modifications. Consistent with the potential regulation of TRIM72 by various oxidative modifications, we found that SNO levels increased at C144 of TRIM72 with ischemic preconditioning. Therefore, to investigate the role of C144 in the regulation of TRIM72 function, we mutated C144 of TRIM72 to a serine residue (TRIM72(C144S)), and expressed either TRIM72(WT) or TRIM72(C144S) in HEK-293 cells, which lack endogenous TRIM72, in order to examine the effect of this mutation on the functional stability of TRIM72 and on cell survival. We hypothesized that SNO of TRIM72 stabilizes the protein, thus allowing for membrane repair and enhanced cell survival. Upon treatment with hydrogen peroxide (H2O2), we found that TRIM72(WT) levels were decreased, but not TRIM72(C144S) and this correlated with increased H2O2-induced cell death in TRIM72(WT) cells. Additionally, we found that treatment with the cardioprotective S-nitrosylating agent S-nitrosoglutathione (GSNO), was able to preserve TRIM72(WT) protein levels and enhance TRIM72(WT)-mediated cell survival, but had no effect on TRIM72(C144S) levels. Consistent with our hypothesis, GSNO was also found to increase SNO levels and inhibit H2O2-induced irreversible oxidation for TRIM72(WT) without affecting TRIM72(C144S). In further support of our hypothesis, GSNO blocked the ischemia/reperfusion-induced decrease in TRIM72 levels and reduced infarct size in a Langendorff-perfused heart model. The results of these studies have important implications for cardioprotection and suggest that SNO of TRIM72 at C144 prevents the oxidation-induced degradation of TRIM72 following oxidative insult, therefore enhancing cardiomyocyte survival. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cyclooxygenase-2 expression and oxidative DNA adducts in murine intestinal adenomas: modification by dietary curcumin and implications for clinical trials.

PubMed

Tunstall, R G; Sharma, R A; Perkins, S; Sale, S; Singh, R; Farmer, P B; Steward, W P; Gescher, A J

2006-02-01

The natural polphenol, curcumin, retards the growth of intestinal adenomas in the Apc(Min+) mouse model of human familial adenomatous polyposis. In other preclinical models, curcumin downregulates the transcription of the enzyme cyclooxygenase-2 (COX-2) and decreases levels of two oxidative DNA adducts, the pyrimidopurinone adduct of deoxyguanosine (M1dG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). We have studied COX-2 protein expression and oxidative DNA adduct levels in intestinal adenoma tissue from Apc(Min+) mice to try and differentiate between curcumin's direct pharmacodynamic effects and indirect effects via its inhibition of adenoma growth. Mice received dietary curcumin (0.2%) for 4 or 14 weeks. COX-2 protein, M1dG and 8-oxo-dG levels were measured by Western blot, immunochemical assay and liquid chromatography-mass spectrometry, respectively. In control Apc(Min+) mice, the levels of all three indices measured in adenoma tissue were significantly higher than levels in normal mucosa. Lifetime administration of curcumin reduced COX-2 expression by 66% (P = 0.01), 8-oxo-dG levels by 24% (P < 0.05) and M1dG levels by 39% (P < 0.005). Short-term feeding did not affect total adenoma number or COX-2 expression, but decreased M1dG levels by 43% (P < 0.01). COX-2 protein levels related to adenoma size. These results demonstrate the utility of measuring these oxidative DNA adduct levels to show direct antioxidant effects of dietary curcumin. The effects of long-term dietary curcumin on COX-2 protein levels appear to reflect retardation of adenoma development.

Allostery in a disordered protein: Oxidative modifications to α-Synuclein act distally to regulate membrane binding

PubMed Central

Sevcsik, Eva; Trexler, Adam J.; Dunn, Joanna M.; Rhoades, Elizabeth

2011-01-01

Both oxidative stress and aggregation of the protein α-synuclein (aS) have been implicated as key factors in the etiology of Parkinson’s disease. Specifically, oxidative modifications to aS disrupt its binding to lipid membranes, an interaction considered critical to its native function. Here we seek to provide a mechanistic explanation for this phenomenon by investigating the effects of oxidative nitration of tyrosine residues on the structure of aS and its interaction with lipid membranes. Membrane binding is mediated by the first ~95 residues of aS. We find that nitration of the single tyrosine (Y39) in this domain disrupts binding due to electrostatic repulsion. Moreover, we observe that nitration of the three tyrosines (Y125/133/136) in the C-terminal domain is equally effective in perturbing binding, an intriguing result given that the C-terminus is not thought to interact directly with membranes. Our investigations show that tyrosine nitration results in a change of the conformational states populated by aS in solution, with the most prominent changes occurring in the C-terminal region. These results lead us to suggest that nitration of Y125/133/136 reduces the membrane binding affinity of aS through allosteric coupling by altering the ensemble of conformational states and depopulating those capable of membrane binding. While allostery is a well-established concept for structured proteins, it has only recently been discussed in the context of disordered proteins. We propose that allosteric regulation through modification of specific residues in, or ligand binding to, the C-terminus may even be a general mechanism for modulating aS function. PMID:21491910

Antisense oligonucleotide against GSK-3β in brain of SAMP8 mice improves learning and memory and decreases oxidative stress: Involvement of transcription factor Nrf2 and implications for Alzheimer disease.

PubMed

Farr, Susan A; Ripley, Jessica L; Sultana, Rukhsana; Zhang, Zhaoshu; Niehoff, Michael L; Platt, Thomas L; Murphy, M Paul; Morley, John E; Kumar, Vijaya; Butterfield, D Allan

2014-02-01

Glycogen synthase kinase (GSK)-3β is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimer's disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aβ), and neurodegeneration. In this study we used 12-month-old SAMP8 mice, an AD model, to examine the effects GSK-3β may cause regarding the cognitive impairment and oxidative stress associated with AD. To suppress the level of GSK-3β, SAMP8 mice were treated with an antisense oligonucleotide (GAO) directed at this kinase. We measured a decreased level of GSK-3β in the cortex of the mice, indicating the success of the antisense treatment. Learning and memory assessments of the SAMP8 mice were tested post-antisense treatment using an aversive T-maze and object recognition test, both of which observably improved. In cortex samples of the SAMP8 mice, decreased levels of protein carbonyl and protein-bound HNE were measured, indicating decreased oxidative stress. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a transcription factor known to increase the level of many antioxidants, including glutathione-S transferase (GST), and is negatively regulated by the activity of GSK-3β. Our results indicated the increased nuclear localization of Nrf2 and level of GST, suggesting the increased activity of the transcription factor as a result of GSK-3β suppression, consistent with the decreased oxidative stress observed. Consistent with the improved learning and memory, and consistent with GSK-3b being a tau kinase, we observed decreased tau phosphorylation in brain of GAO-treated SAMP8 mice compared to that of RAO-treated SAMP8 mice. Lastly, we examined the ability of GAO to cross the blood-brain barrier and determined it to be possible. The results presented in this study demonstrate that reducing GSK-3 with a phosphorothionated antisense against GSK-3 improves learning and memory, reduces oxidative stress, possibly coincident with increased levels of the antioxidant transcriptional activity of Nrf2, and decreases tau phosphorylation. Our study supports the notion of GAO as a possible treatment for AD. Copyright © 2013 Elsevier Inc. All rights reserved.

Capturing Labile Sulfenamide and Sulfinamide Serum Albumin Adducts of Carcinogenic Arylamines by Chemical Oxidation

PubMed Central

Peng, Lijuan; Turesky, Robert J.

2013-01-01

Aromatic amines and heterocyclic aromatic amines (HAAs) are a class of structurally related carcinogens that are formed during the combustion of tobacco or during the high temperature cooking of meats. These procarcinogens undergo metabolic activation by N-oxidation of the exocyclic amine group to produce N-hydroxylated metabolites, which are critical intermediates implicated in toxicity and DNA damage. The arylhydroxylamines and their oxidized arylnitroso derivatives can also react with cysteine (Cys) residues of glutathione or proteins to form, respectively, sulfenamide and sulfinamide adducts. However, sulfur-nitrogen linked adducted proteins are often difficult to detect because they are unstable and undergo hydrolysis during proteolytic digestion. Synthetic N-oxidized intermediates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic HAA produced in cooked meats, and 4-aminobiphenyl, a carcinogenic aromatic amine present in tobacco smoke were reacted with human serum albumin (SA) and formed labile sulfenamide or sulfinamide adducts at the Cys34 residue. Oxidation of the carcinogen-modified SA with m-chloroperoxybenzoic acid (m-CPBA) produced the arylsulfonamide adducts, which were stable to heat and the chemical reduction conditions employed to denature SA. The sulfonamide adducts of PhIP and 4-ABP were identified, by liquid chromatography/mass spectrometry, in proteolytic digests of denatured SA. Thus, selective oxidation of arylamine-modified SA produces stable arylsulfonamide-SA adducts, which may serve as biomarkers of these tobacco and dietary carcinogens. PMID:23240913

APE1 promotes antioxidant capacity by regulating Nrf-2 function through a redox-dependent mechanism.

PubMed

Shan, Jin-Lu; He, Hai-Tao; Li, Meng-Xia; Zhu, Jian-Wu; Cheng, Yi; Hu, Nan; Wang, Ge; Wang, Dong; Yang, Xue-Qin; He, Yong; Xiao, Hua-Liang; Tong, Wei-Dong; Yang, Zhen-Zhou

2015-01-01

APE1 is a multifunctional protein that has recently been implicated in protecting cells from oxidative stress. In the current study, we confirmed that APE1׳s effect on cellular antioxidant capacity is related to its redox activity through the use of an APE1 functional mutant, and we investigated the mechanism through which this multifunctional protein affects the function of the transcription factor Nrf-2 in regulating oxidative stress-induced genes. Using a pair of mutants for both the redox activity and the acetylation-regulated activity of APE1, in vitro assays showed that the redox activity of APE1 is crucial for its nuclear association with Nrf-2 and subsequent activation of Nrf-2׳s transcription of several downstream genes during oxidative challenge. Important oxidative stress genes are affected by APE1 redox activity, including Hmox1, Gstm1, and Txnrd1. In addition, utilizing human non-small-cell lung cancer sample tissue as well as a nude mouse xenograft model, we determined that APE1 expression levels are inversely correlated to oxidative stress in vivo. These findings indicated that interference with these crucial functions of APE1 shows promise in preventing resistance to certain radiotherapies and that further research is necessary to understand APE1׳s complex roles in regulating both the basal redox status and the oxidative stress state of the cellular environment. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

TXNIP regulates myocardial fatty acid oxidation via miR-33a signaling.

PubMed

Chen, Junqin; Young, Martin E; Chatham, John C; Crossman, David K; Dell'Italia, Louis J; Shalev, Anath

2016-07-01

Myocardial fatty acid β-oxidation is critical for the maintenance of energy homeostasis and contractile function in the heart, but its regulation is still not fully understood. While thioredoxin-interacting protein (TXNIP) has recently been implicated in cardiac metabolism and mitochondrial function, its effects on β-oxidation have remained unexplored. Using a new cardiomyocyte-specific TXNIP knockout mouse and working heart perfusion studies, as well as loss- and gain-of-function experiments in rat H9C2 and human AC16 cardiomyocytes, we discovered that TXNIP deficiency promotes myocardial β-oxidation via signaling through a specific microRNA, miR-33a. TXNIP deficiency leads to increased binding of nuclear factor Y (NFYA) to the sterol regulatory element binding protein 2 (SREBP2) promoter, resulting in transcriptional inhibition of SREBP2 and its intronic miR-33a. This allows for increased translation of the miR-33a target genes and β-oxidation-promoting enzymes, carnitine octanoyl transferase (CROT), carnitine palmitoyl transferase 1 (CPT1), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase-β (HADHB), and AMPKα and is associated with an increase in phospho-AMPKα and phosphorylation/inactivation of acetyl-CoA-carboxylase. Thus, we have identified a novel TXNIP-NFYA-SREBP2/miR-33a-AMPKα/CROT/CPT1/HADHB pathway that is conserved in mouse, rat, and human cardiomyocytes and regulates myocardial β-oxidation. Copyright © 2016 the American Physiological Society.

Free radicals: properties, sources, targets, and their implication in various diseases.

PubMed

Phaniendra, Alugoju; Jestadi, Dinesh Babu; Periyasamy, Latha

2015-01-01

Free radicals and other oxidants have gained importance in the field of biology due to their central role in various physiological conditions as well as their implication in a diverse range of diseases. The free radicals, both the reactive oxygen species (ROS) and reactive nitrogen species (RNS), are derived from both endogenous sources (mitochondria, peroxisomes, endoplasmic reticulum, phagocytic cells etc.) and exogenous sources (pollution, alcohol, tobacco smoke, heavy metals, transition metals, industrial solvents, pesticides, certain drugs like halothane, paracetamol, and radiation). Free radicals can adversely affect various important classes of biological molecules such as nucleic acids, lipids, and proteins, thereby altering the normal redox status leading to increased oxidative stress. The free radicals induced oxidative stress has been reported to be involved in several diseased conditions such as diabetes mellitus, neurodegenerative disorders (Parkinson's disease-PD, Alzheimer's disease-AD and Multiple sclerosis-MS), cardiovascular diseases (atherosclerosis and hypertension), respiratory diseases (asthma), cataract development, rheumatoid arthritis and in various cancers (colorectal, prostate, breast, lung, bladder cancers). This review deals with chemistry, formation and sources, and molecular targets of free radicals and it provides a brief overview on the pathogenesis of various diseased conditions caused by ROS/RNS.

Effects of short-term Western diet on cerebral oxidative stress and diabetes related factors in APP x PS1 knock-in mice.

PubMed

Studzinski, Christa M; Li, Feng; Bruce-Keller, Annadora J; Fernandez-Kim, Sun Ok; Zhang, Le; Weidner, Adam M; Markesbery, William R; Murphy, M Paul; Keller, Jeffrey N

2009-02-01

A chronic high fat Western diet (WD) promotes a variety of morbidity factors although experimental evidence for short-term WD mediating brain dysfunction remains to be elucidated. The amyloid precursor protein and presenilin-1 (APP x PS1) knock-in mouse model has been demonstrated to recapitulate some key features of Alzheimer's disease pathology, including amyloid-beta (Abeta) pathogenesis. In this study, we placed 1-month-old APP x PS1 mice and non-transgenic littermates on a WD for 4 weeks. The WD resulted in a significant elevation in protein oxidation and lipid peroxidation in the brain of APP x PS1 mice relative to non-transgenic littermates, which occurred in the absence of increased Abeta levels. Altered adipokine levels were also observed in APP x PS1 mice placed on a short-term WD, relative to non-transgenic littermates. Taken together, these data indicate that short-term WD is sufficient to selectively promote cerebral oxidative stress and metabolic disturbances in APP x PS1 knock-in mice, with increased oxidative stress preceding alterations in Abeta. These data have important implications for understanding how WD may potentially contribute to brain dysfunction and the development of neurodegenerative disorders such as Alzheimer's disease.

Autophagy as a melanocytic self-defense mechanism.

PubMed

Setaluri, Vijayasaradhi

2015-05-01

Defects in autophagy have implications for melanocyte survival and manifestations of skin pigmentary disorders. Zhang et al. (2015) show that mouse melanocytes lacking the autophagy protein Atg7 undergo premature senescence in vitro and accumulate products of oxidative damage, despite activation of the redox response. Interestingly, contrary to previous findings, the melanocyte-specific deficiency in autophagy did not cause major defects in melanosome biogenesis, nor did it produce visually striking changes in mouse coat color.

Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria.

PubMed

Marcus, Dana; Lichtenstein, Michal; Cohen, Natali; Hadad, Rita; Erlich-Hadad, Tal; Greif, Hagar; Lorberboum-Galski, Haya

2016-12-01

Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cigarette smoke inhibits macrophage sensing of Gram-negative bacteria and lipopolysaccharide: relative roles of nicotine and oxidant stress

PubMed Central

McMaster, S K; Paul-Clark, M J; Walters, M; Fleet, M; Anandarajah, J; Sriskandan, S; Mitchell, J A

2007-01-01

Background and purpose: Smoking cigarettes is a major risk factor for the development of cardiovascular and respiratory disease. Moreover, smokers are more prone to infections. This has been associated with a suppression of the immune system by smoke. However, it is not clear how cigarette smoke affects the ability of immune cells to sense pathogens. Cigarette smoke contains a large number of molecules which may mediate responses on immune cells and of these, nicotine and oxidants have both been identified as inhibitory for the sensing of bacterial lipopolysaccharide (LPS). Nitric oxide synthase (NOS) and tumour necrosis factor (TNF)-α are both induced in macrophages on stimulation with Gram negative bacteria or LPS. Experimental approach: We used murine macrophages stimulated with whole heat-killed bacteria or LPS. We measured output of NO (as nitrite) and TNFα, NOS protein by Western blotting and cellular oxidant stress. Key results: Cigarette smoke extract suppressed the ability of murine macrophages to release NO, but not TNFα in response to whole bacteria. Cigarette smoke extract also inhibited nitric oxide synthase II protein expression in response to LPS. The effects of cigarette smoke extract on nitrite formation stimulated by LPS were unaffected by inhibition of nicotinic receptors with α-bungarotoxin (100 units ml−1). However, the effects of cigarette smoke extract on LPS-induced nitrite formation were mimicked by hydrogen peroxide and reversed by the anti-oxidants N-acetyl cysteine and glutathione. Conclusions and implications: We suggest that cigarette smoke exerts its immunosuppressive effects through an oxidant-dependent and not a nicotine-dependent mechanism. PMID:18059323

Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets

PubMed Central

Farazi, Thalia A.; Leonhardt, Carl S.; Mukherjee, Neelanjan; Mihailovic, Aleksandra; Li, Song; Max, Klaas E.A.; Meyer, Cindy; Yamaji, Masashi; Cekan, Pavol; Jacobs, Nicholas C.; Gerstberger, Stefanie; Bognanni, Claudia; Larsson, Erik; Ohler, Uwe; Tuschl, Thomas

2014-01-01

Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed. PMID:24860013

Markers of Oxidant Stress that are Clinically Relevant in Aging and Age-related Disease

PubMed Central

Jacob, Kimberly D.; Hooten, Nicole Noren; Trzeciak, Andrzej R.; Evans, Michele K.

2013-01-01

Despite the long held hypothesis that oxidant stress results in accumulated oxidative damage to cellular macromolecules and subsequently to aging and age-related chronic disease, it has been difficult to consistently define and specifically identify markers of oxidant stress that are consistently and directly linked to age and disease status. Inflammation because it is also linked to oxidant stress, aging, and chronic disease also plays an important role in understanding the clinical implications of oxidant stress and relevant markers. Much attention has focused on identifying specific markers of oxidative stress and inflammation that could be measured in easily accessible tissues and fluids (lymphocytes, plasma, serum). The purpose of this review is to discuss markers of oxidant stress used in the field as biomarkers of aging and age-related diseases, highlighting differences observed by race when data is available. We highlight DNA, RNA, protein, and lipid oxidation as measures of oxidative stress, as well as other well-characterized markers of oxidative damage and inflammation and discuss their strengths and limitations. We present the current state of the literature reporting use of these markers in studies of human cohorts in relation to age and age-related disease and also with a special emphasis on differences observed by race when relevant. PMID:23428415

The role of vitamin D in malaria.

PubMed

Lương, Khanh Vinh Quốc; Nguyễn, Lan Thi Hoàng

2015-01-15

An abnormal calcium-parathyroid hormone (PTH)-vitamin D axis has been reported in patients with malaria infection. A role for vitamin D in malaria has been suggested by many studies. Genetic studies have identified numerous factors that link vitamin D to malaria, including human leukocyte antigen genes, toll-like receptors, heme oxygenase-1, angiopoietin-2, cytotoxic T lymphocyte antigen-4, nucleotide-binding oligomerization domain-like receptors, and Bcl-2. Vitamin D has also been implicated in malaria via its effects on the Bacillus Calmette-Guerin (BCG) vaccine, matrix metalloproteinases, mitogen-activated protein kinase pathways, prostaglandins, reactive oxidative species, and nitric oxide synthase. Vitamin D may be important in malaria; therefore, additional research on its role in malaria is needed.

Structural characterization of the P1+ intermediate state of the P-cluster of nitrogenase.

PubMed

Keable, Stephen M; Zadvornyy, Oleg A; Johnson, Lewis E; Ginovska, Bojana; Rasmussen, Andrew J; Danyal, Karamatullah; Eilers, Brian J; Prussia, Gregory A; LeVan, Axl X; Raugei, Simone; Seefeldt, Lance C; Peters, John W

2018-05-02

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N 2 ) to ammonia (NH 3 ) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N 2 is reduced to NH 3 The [8Fe-7S] P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox dependent structural changes and that the transition from the all-ferrous resting (P N ) state to the two electron oxidized P 2+ state is accompanied by protein serince hydroxyl and backbone amide ligation to Fe. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P 2+ , P 1+ , and P N ) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination implicating that the P 1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P 2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism.

PubMed

Auernik, Kathryne S; Maezato, Yukari; Blum, Paul H; Kelly, Robert M

2008-02-01

Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, approximately 2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized.

The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon Metallosphaera sedula Provides Insights into Bioleaching-Associated Metabolism▿ †

PubMed Central

Auernik, Kathryne S.; Maezato, Yukari; Blum, Paul H.; Kelly, Robert M.

2008-01-01

Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ∼2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized. PMID:18083856

Role of Sirt1 during the ageing process: relevance to protection of synapses in the brain.

PubMed

Godoy, Juan A; Zolezzi, Juan M; Braidy, Nady; Inestrosa, Nibaldo C

2014-12-01

Ageing is a stochastic process associated with a progressive decline in physiological functions which predispose to the pathogenesis of several neurodegenerative diseases. The intrinsic complexity of ageing remains a significant challenge to understand the cause of this natural phenomenon. At the molecular level, ageing is thought to be characterized by the accumulation of chronic oxidative damage to lipids, proteins and nucleic acids caused by free radicals. Increased oxidative stress and misfolded protein formations, combined with impaired compensatory mechanisms, may promote neurodegenerative disorders with age. Nutritional modulation through calorie restriction has been shown to be effective as an anti-ageing factor, promoting longevity and protecting against neurodegenerative pathology in yeast, nematodes and murine models. Calorie restriction increases the intracellular levels of the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD(+)), a co-substrate for the sirtuin 1 (Sirt1, silent mating-type information regulator 2 homolog 1) activity and a cofactor for oxidative phosphorylation and ATP synthesis. Promotion of intracellular NAD(+) anabolism is speculated to induce neuroprotective effects against amyloid-β-peptide (Aβ) toxicity in some models for Alzheimer's disease (AD). The NAD(+)-dependent histone deacetylase, Sirt1, has been implicated in the ageing process. Sirt1 serves as a deacetylase for numerous proteins involved in several cellular pathways, including stress response and apoptosis, and plays a protective role in neurodegenerative disorders, such as AD.

Ceruloplasmin (ferroxidase) oxidizes hydroxylamine probes: deceptive implications for free radical detection.

PubMed

Ganini, Douglas; Canistro, Donatella; Jiang, JinJie; Jang, JinJie; Stadler, Krisztian; Mason, Ronald P; Kadiiska, Maria B

2012-10-01

Ceruloplasmin (ferroxidase) is a copper-binding protein known to promote Fe(2+) oxidation in plasma of mammals. In addition to its classical ferroxidase activity, ceruloplasmin is known to catalyze the oxidation of various substrates, such as amines and catechols. Assays based on cyclic hydroxylamine oxidation are used to quantify and detect free radicals in biological samples ex vivo and in vitro. We show here that human ceruloplasmin promotes the oxidation of the cyclic hydroxylamine 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) and related probes in Chelex-treated phosphate buffer and rat serum. The reaction is suppressed by the metal chelators DTPA, EDTA, and desferal, whereas heparin and bathocuproine have no effect. Catalase or superoxide dismutase additions do not interfere with the CPH-oxidation yield, demonstrating that oxygen-derived free radicals are not involved in the CPH oxidation mediated by ceruloplasmin. Plasma samples immunodepleted of ceruloplasmin have lower levels of CPH oxidation, which confirms the role of ceruloplasmin (ferroxidase) as a biological oxidizing agent of cyclic hydroxylamines. In conclusion, we show that the ferroxidase activity of ceruloplasmin is a possible biological source of artifacts in the cyclic hydroxylamine-oxidation assay used for reactive oxygen species detection and quantification. Published by Elsevier Inc.

Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis; Implications for Vaccine Development

PubMed Central

van de Waterbeemd, Bas; Zomer, Gijsbert; van den IJssel, Jan; van Keulen, Lonneke; Eppink, Michel H.; van der Ley, Peter; van der Pol, Leo A.

2013-01-01

Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis. PMID:23372704

Skeletal muscle action of estrogen receptor α is critical for the maintenance of mitochondrial function and metabolic homeostasis in females

PubMed Central

Ribas, Vicent; Drew, Brian G.; Zhou, Zhenqi; Phun, Jennifer; Kalajian, Nareg Y.; Soleymani, Teo; Daraei, Pedram; Widjaja, Kevin; Wanagat, Jonathan; de Aguiar Vallim, Thomas Q.; Fluitt, Amy H.; Bensinger, Steven; Le, Thuc; Radu, Caius; Whitelegge, Julian P.; Beaven, Simon W.; Tontonoz, Peter; Lusis, Aldons J.; Parks, Brian W.; Vergnes, Laurent; Reue, Karen; Singh, Harpreet; Bopassa, Jean C.; Toro, Ligia; Stefani, Enrico; Watt, Matthew J.; Schenk, Simon; Akerstrom, Thorbjorn; Kelly, Meghan; Pedersen, Bente K.; Hewitt, Sylvia C.; Korach, Kenneth S.; Hevener, Andrea L.

2016-01-01

Impaired estrogen receptor α(ERα) action promotes obesity and metabolic dysfunction in humans and mice; however, the mechanisms underlying these phenotypes remain unknown. Considering that skeletal muscle is a primary tissue responsible for glucose disposal and oxidative metabolism, we established that reduced ERαexpression in muscle is associated with glucose intolerance and adiposity in women and female mice. To test this relationship, we generated muscle-specific ERαknockout (MERKO) mice. Impaired glucose homeostasis and increased adiposity were paralleled by diminished muscle oxidative metabolism and bioactive lipid accumulation in MERKO mice. Aberrant mitochondrial morphology, overproduction of reactive oxygen species, and impairment in basal and stress-induced mitochondrial fission dynamics, driven by imbalanced protein kinase A–regulator of calcineurin 1–calcineurin signaling through dynamin-related protein 1, tracked with reduced oxidative metabolism in MERKO muscle. Although muscle mitochondrial DNA (mtDNA) abundance was similar between the genotypes, ERαdeficiency diminished mtDNA turnover by a balanced reduction in mtDNA replication and degradation. Our findings indicate the retention of dysfunctional mitochondria in MERKO muscle and implicate ERαin the preservation of mitochondrial health and insulin sensitivity as a defense against metabolic disease in women. PMID:27075628

Solving Kinetic Equations for the Laser Flash Photolysis Experiment on Nitric Oxide Synthases: Effect of Conformational Dynamics on the Interdomain Electron Transfer.

PubMed

Astashkin, Andrei V; Feng, Changjian

2015-11-12

The production of nitric oxide by the nitric oxide synthase (NOS) enzyme depends on the interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme domains. Although the rate of this IET has been measured by laser flash photolysis (LFP) for various NOS proteins, no rigorous analysis of the relevant kinetic equations was performed so far. In this work, we provide an analytical solution of the kinetic equations underlying the LFP approach. The derived expressions reveal that the bulk IET rate is significantly affected by the conformational dynamics that determines the formation and dissociation rates of the docking complex between the FMN and heme domains. We show that in order to informatively study the electron transfer across the NOS enzyme, LFP should be used in combination with other spectroscopic methods that could directly probe the docking equilibrium and the conformational change rate constants. The implications of the obtained analytical expressions for the interpretation of the LFP results from various native and modified NOS proteins are discussed. The mathematical formulas derived in this work should also be applicable for interpreting the IET kinetics in other modular redox enzymes.

H2O2 Regulates Lung Epithelial Sodium Channel (ENaC) via Ubiquitin-like Protein Nedd8

PubMed Central

Downs, Charles A.; Kumar, Amrita; Kreiner, Lisa H.; Johnson, Nicholle M.; Helms, My N.

2013-01-01

Redundancies in both the ubiquitin and epithelial sodium transport pathways allude to their importance of proteolytic degradation and ion transport in maintaining normal cell function. The classical pathway implicated in ubiquitination of the epithelial sodium channel (ENaC) involves Nedd4-2 regulation of sodium channel subunit expression and has been studied extensively studied. However, less attention has been given to the role of the ubiquitin-like protein Nedd8. Here we show that Nedd8 plays an important role in the ubiquitination of ENaC in alveolar epithelial cells. We report that the Nedd8 pathway is redox-sensitive and that under oxidizing conditions Nedd8 conjugation to Cullin-1 is attenuated, resulting in greater surface expression of α-ENaC. This observation was confirmed in our electrophysiology studies in which we inhibited Nedd8-activating enzyme using MLN4924 (a specific Nedd8-activating enzyme inhibitor) and observed a marked increase in ENaC activity (measured as the product of the number of channels (N) and the open probability (Po) of a channel). These results suggest that ubiquitination of lung ENaC is redox-sensitive and may have significant implications for our understanding of the role of ENaC in pulmonary conditions where oxidative stress occurs, such as pulmonary edema and acute lung injury. PMID:23362276

Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages

PubMed Central

Vogt, Carmen; Pernemalm, Maria; Kohonen, Pekka; Laurent, Sophie; Hultenby, Kjell; Vahter, Marie; Lehtiö, Janne; Toprak, Muhammet S.; Fadeel, Bengt

2015-01-01

Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated versus dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs. PMID:26444829

Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages.

PubMed

Vogt, Carmen; Pernemalm, Maria; Kohonen, Pekka; Laurent, Sophie; Hultenby, Kjell; Vahter, Marie; Lehtiö, Janne; Toprak, Muhammet S; Fadeel, Bengt

2015-01-01

Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated versus dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs.

Gestational diabetes is characterized by reduced mitochondrial protein expression and altered calcium signaling proteins in skeletal muscle.

PubMed

Boyle, Kristen E; Hwang, Hyonson; Janssen, Rachel C; DeVente, James M; Barbour, Linda A; Hernandez, Teri L; Mandarino, Lawrence J; Lappas, Martha; Friedman, Jacob E

2014-01-01

The rising prevalence of gestational diabetes mellitus (GDM) affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM) and obese pregnant women with normal glucose tolerance (ONGT). Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I) subunits (NDUFS3, NDUFV2) and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, α1-syntrophin, annexin A4) in OGDM (n = 6) vs. ONGT (n = 6). Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (-60-75%) in the OGDM (n = 8) compared with ONGT (n = 10) subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum.

Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia

PubMed Central

Bakker, Astrid D.; Hogervorst, Jolanda M. A.; Nolte, Peter A.; Klein-Nulend, Jenneke

2017-01-01

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation. PMID:28166273

New concepts of microbial treatment processes for the nitrogen removal: effect of protein and amino acids degradation.

PubMed

González-Martínez, Alejandro; Calderón, Kadiya; González-López, Jesús

2016-05-01

High concentrations of proteins and amino acids can be found in wastewater and wastewater stream produced in anaerobic digesters, having shown that amino acids could persist over different managements for nitrogen removal affecting the nitrogen removal processes. Nitrogen removal is completely necessary because of their implications and the significant adverse environmental impact of ammonium such as eutrophication and toxicity to aquatic life on the receiving bodies. In the last decade, the treatment of effluents with high ammonium concentration through anammox-based bioprocesses has been enhanced because these biotechnologies are cheaper and more environmentally friendly than conventional technologies. However, it has been shown that the presence of important amounts of proteins and amino acids in the effluents seriously affects the microbial autotrophic consortia leading to important losses in terms of ammonium oxidation efficiency. Particularly the presence of sulfur amino acids such as methionine and cysteine has been reported to drastically decrease the autotrophic denitrification processes as well as affect the microbial community structure promoting the decline of ammonium oxidizing bacteria in favor of other phylotypes. In this context we discuss that new biotechnological processes that improve the degradation of protein and amino acids must be considered as a priority to increase the performance of the autotrophic denitrification biotechnologies.

AMP-activated protein kinase: Role in metabolism and therapeutic implications.

PubMed

Schimmack, Greg; Defronzo, Ralph A; Musi, Nicolas

2006-11-01

AMP-activated protein kinase (AMPK) is an enzyme that works as a fuel gauge which becomes activated in situations of energy consumption. AMPK functions to restore cellular ATP levels by modifying diverse metabolic and cellular pathways. In the skeletal muscle, AMPK is activated during exercise and is involved in contraction-stimulated glucose transport and fatty acid oxidation. In the heart, AMPK activity increases during ischaemia and functions to sustain ATP, cardiac function and myocardial viability. In the liver, AMPK inhibits the production of glucose, cholesterol and triglycerides and stimulates fatty acid oxidation. Recent studies have shown that AMPK is involved in the mechanism of action of metformin and thiazolidinediones, and the adipocytokines leptin and adiponectin. These data, along with evidence that pharmacological activation of AMPK in vivo improves blood glucose homeostasis, cholesterol concentrations and blood pressure in insulin-resistant rodents, make this enzyme an attractive pharmacological target for the treatment of type 2 diabetes, ischaemic heart disease and other metabolic diseases.

Prohibitin is associated with antioxidative protection in hypoxia/reoxygenation-induced renal tubular epithelial cell injury

NASA Astrophysics Data System (ADS)

Zhou, Tian-Biao; Qin, Yuan-Han; Lei, Feng-Ying; Huang, Wei-Fang; Drummen, Gregor P. C.

2013-11-01

Prohibitin is an evolutionary conserved and pleiotropic protein that has been implicated in various cellular functions, including proliferation, tumour suppression, apoptosis, transcription, and mitochondrial protein folding. We recently demonstrated that prohibitin downregulation results in increased renal interstitial fibrosis. Here we investigated the role of oxidative stress and prohibitin expression in a hypoxia/reoxygenation injury system in renal tubular epithelial cells with lentivirus-based delivery vectors to knockdown or overexpress prohibitin. Our results show that increased prohibitin expression was negatively correlated with reactive oxygen species, malon dialdehyde, transforming-growth-factor-β1, collagen-IV, fibronectin, and apoptosis (r = -0.895, -0.764, -0.798, -0.826, -0.817, -0.735 each P < 0.01), but positively correlated with superoxide dismutase, glutathione and mitochondrial membrane potential (r = 0.807, 0.815, 0.739; each P < 0.01). We postulate that prohibitin acts as a positive regulator of mechanisms that counteract oxidative stress and extracellular matrix accumulation and therefore has an antioxidative effect.

Exploring the molecular mechanism of trimethylamine-N-oxide's ability to counteract the protein denaturing effects of urea.

PubMed

Sarma, Rahul; Paul, Sandip

2013-05-09

Protein denaturation in highly concentrated urea solution is a well-known phenomenon. The counteracting effect of a naturally occurring osmolyte, trimethylamine-N-oxide (TMAO), against urea-conferred protein denaturation is also well-established. However, what is largely unknown is the mechanism by which TMAO counteracts this denaturation. To provide a molecular level understanding of how TMAO protects proteins in highly concentrated urea solution, we report here the structural, energetic, and dynamical properties of N-methylacetamide (NMA) solutions that also contain urea and/or TMAO. The solute NMA is of interest mainly because it contains the peptide linkage in addition to hydrophobic sites and represents the typical solvent-exposed state of proteins. Molecular dynamics computer simulation technique is employed in this study. Analysis of solvation characteristics reveals dehydration of NMA and reduction in hydrogen bond number between NMA oxygen and water upon addition of TMAO. The effect of TMAO on NMA-urea interaction is found to be insignificant. Because TMAO cannot donate its hydrogen to NMA oxygen, the total number of hydrogen bonds formed by NMA oxygen with solution species decreases in the presence of TMAO. In solution, TMAO is found to interact strongly with water and urea. Solvation of TMAO makes the water hydrogen bonding network relatively stronger and reduces relaxation of urea-water hydrogen bonds. Implications of these results for counteracting mechanism of TMAO are discussed.

Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

PubMed Central

He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

2016-01-01

Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease. PMID:27488468

Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

NASA Astrophysics Data System (ADS)

He, Fengping; Xu, Xin; Yuan, Shuguo; Tan, Liangqiu; Gao, Lingjun; Ma, Shaochun; Zhang, Shebin; Ma, Zhanzhong; Jiang, Wei; Liu, Fenglian; Chen, Baofeng; Zhang, Beibei; Pang, Jungang; Huang, Xiuyan; Weng, Jiaqiang

2016-08-01

Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease.

Dependence of Golgi apparatus integrity on nitric oxide in vascular cells: implications in pulmonary arterial hypertension

PubMed Central

Lee, Jason E.; Patel, Kirit; Almodóvar, Sharilyn; Tuder, Rubin M.; Flores, Sonia C.

2011-01-01

Although reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), its consequences on organellar structure and function within vascular cells is largely unexplored. We investigated the effect of reduced NO on the structure of the Golgi apparatus as assayed by giantin or GM130 immunofluorescence in human pulmonary arterial endothelial (HPAECs) and smooth muscle (HPASMCs) cells, bovine PAECs, and human EA.hy926 endothelial cells. Golgi structure was also investigated in cells in tissue sections of pulmonary vascular lesions in idiopathic PAH (IPAH) and in macaques infected with a chimeric simian immunodeficiency virus containing the human immunodeficiency virus (HIV)-nef gene (SHIV-nef) with subcellular three-dimensional (3D) immunoimaging. Compounds with NO scavenging activity including 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), methylene blue, N-acetylcysteine, and hemoglobin markedly fragmented the Golgi in all cell types evaluated as did monocrotaline pyrrole, while LY-83583, sildenafil, fasudil, Y-27632, Tiron, Tempol, or H2O2 did not. Golgi fragmentation by NO scavengers was inhibited by diethylamine NONOate, was evident in HPAECs after selective knockdown of endothelial nitric oxide synthase using small interfering RNA (siRNA), was independent of microtubule organization, required the GTPase dynamin 2, and was accompanied by depletion of α-soluble N-ethylmaleimide-sensitive factor (NSF) acceptor protein (α-SNAP) from Golgi membranes and codispersal of the SNAP receptor (SNARE) Vti1a with giantin. Golgi fragmentation was confirmed in endothelial and smooth muscle cells in pulmonary arterial lesions in IPAH and the SHIV-nef-infected macaque with subcellular 3D immunoimaging. In SHIV-nef-infected macaques Golgi fragmentation was observed in cells containing HIV-nef-bearing endosomes. The observed Golgi fragmentation suggests that NO plays a significant role in modulating global protein trafficking patterns that contribute to changes in the cell surface landscape and functional signaling in vascular cells. PMID:21217069

Zinc stimulates glucose oxidation and glycemic control by modulating the insulin signaling pathway in human and mouse skeletal muscle cell lines.

PubMed

Norouzi, Shaghayegh; Adulcikas, John; Sohal, Sukhwinder Singh; Myers, Stephen

2018-01-01

Zinc is a metal ion that is an essential cell signaling molecule. Highlighting this, zinc is an insulin mimetic, activating cellular pathways that regulate cellular homeostasis and physiological responses. Previous studies have linked dysfunctional zinc signaling with several disease states including cancer, obesity, cardiovascular disease and type 2 diabetes. The present study evaluated the insulin-like effects of zinc on cell signaling molecules including tyrosine, PRSA40, Akt, ERK1/2, SHP-2, GSK-3β and p38, and glucose oxidation in human and mouse skeletal muscle cells. Insulin and zinc independently led to the phosphorylation of these proteins over a 60-minute time course in both mouse and human skeletal muscle cells. Similarly, utilizing a protein array we identified that zinc could active the phosphorylation of p38, ERK1/2 and GSK-3B in human and ERK1/2 and GSK-3B in mouse skeletal muscle cells. Glucose oxidation assays were performed on skeletal muscle cells treated with insulin, zinc, or a combination of both and resulted in a significant induction of glucose consumption in mouse (p<0.01) and human (p<0.05) skeletal muscle cells when treated with zinc alone. Insulin, as expected, increased glucose oxidation in mouse (p<0.001) and human (0.001) skeletal muscle cells, however the combination of zinc and insulin did not augment glucose consumption in these cells. Zinc acts as an insulin mimetic, activating key molecules implicated in cell signaling to maintain glucose homeostasis in mouse and human skeletal muscle cells. Zinc is an important metal ion implicated in several biological processes. The role of zinc as an insulin memetic in activating key signaling molecules involved in glucose homeostasis could provide opportunities to utilize this ion therapeutically in treating disorders associated with dysfunctional zinc signaling.

Redox implications of AMPK-mediated signal transduction beyond energetic clues.

PubMed

Cardaci, Simone; Filomeni, Giuseppe; Ciriolo, Maria Rosa

2012-05-01

Since the discovery of AMP-dependent protein kinase (AMPK), its fundamental role in regulating metabolic pathways and the molecular mechanism underlying the regulation of its activity by adenine nucleotides has been widely studied. AMPK is not only an energy-responsive enzyme, but it also senses redox signals. This review aims at recapitulating the recent lines of evidence that demonstrate the responsiveness of this kinase to metabolic and nitroxidative imbalance, thus providing new insights into the intimate networks of redox-based signals upstream of AMPK. In particular, we discuss its well-recognized activation downstream of mitochondrial dysfunction, debate the recent findings that AMPK is directly targeted by pro-oxidant species, and question alternative redox pathways that allow AMPK to be included into the large class of redox-sensing proteins. The possible therapeutic implications of the role of AMPK in redox-associated pathologies, such as cancer and neurodegeneration, are also discussed in light of recent advances that suggest a role for AMPK in the tuning of redox-dependent processes, such as apoptosis and autophagy.

Autoxidation products of both carbohydrates and lipids are increased in uremic plasma: is there oxidative stress in uremia?

PubMed

Miyata, T; Fu, M X; Kurokawa, K; van Ypersele de Strihou, C; Thorpe, S R; Baynes, J W

1998-10-01

Advanced glycation end products (AGEs), formed by non-enzymatic glycation and oxidation (glycoxidation) reactions, have been implicated in the pathogenesis of several diseases, including normoglycemic uremia. AGE research in uremia has focused on the accumulation of carbohydrate-derived adducts generated by the Maillard reaction. Recent studies, however, have demonstrated that one AGE, the glycoxidation product carboxymethyllysine (CML), could be derived not only from carbohydrates but also from oxidation of polyunsaturated fatty acids in vitro, raising the possibility that both carbohydrate and lipid autoxidation might be increased in uremia. To address this hypothesis, we applied gas chromatography-mass spectrometry and high performance liquid chromatography to measure protein adducts formed in uremic plasma by reactions between carbonyl compounds and protein amino groups: pentosidine derived from carbohydrate-derived carbonyls, malondialdehyde (MDA)-lysine derived from lipid-derived carbonyls, and CML originating possibly from both sources. All three adducts were elevated in uremic plasma. Plasma CML levels were mainly (>95%) albumin bound. Their levels were not correlated with fructoselysine levels and were similar in diabetic and non-diabetic patients on hemodialysis, indicating that their increase was not driven by glucose. Pentosidine and MDA-lysine were also increased in plasma to the same extent in diabetic and non-diabetic hemodialysis patients. Statistical analysis indicated that plasma levels of CML correlated weakly (P < 0.05) with those of pentosidine and MDA-lysine, but that pentosidine and MDA-lysine varied independently (P > 0.5). These data suggest that the increased levels of AGEs in blood, and probably in tissues, reported in uremia implicate a broad derangement in non-enzymatic biochemistry involving alterations in autoxidation of both carbohydrates and lipids.

Vitamin C prophylaxis promotes oxidative lipid damage during surgical ischemia-reperfusion.

PubMed

Bailey, Damian M; Raman, Sudarsanam; McEneny, Jane; Young, Ian S; Parham, Kelly L; Hullin, David A; Davies, Bruce; McKeeman, Gareth; McCord, Joe M; Lewis, Michael H

2006-02-15

Reactive oxygen species (ROS) have been implicated in the cellular membrane damage and postoperative morbidity associated with obligatory ischemia-reperfusion (I-R) during vascular surgery. Thus, a clinical study was undertaken to evaluate the effects of ascorbate prophylaxis on ROS exchange kinetics in 22 patients scheduled for elective abdominal aortic aneurysm (AAA) or infra-inguinal bypass (IIB) repair. Patients were assigned double-blind to receive intravenous sodium ascorbate (2 g vitamin C, n=10) or placebo (0.9% saline, n=12) administered 2 h prior to surgery. Blood samples were obtained from the arterial and venous circulation proximal to the respective sites of surgical repair (local) and from an antecubital vein (peripheral) during cross-clamping (ischemia) and within 60 s of clamp release (reperfusion). Ascorbate supplementation increased the venoarterial concentration difference (v-adiff) of lipid hydroperoxides (LH), interleukin (IL)-6 and vascular endothelial growth factor (VEGF) protein during ischemia. This increased the peripheral concentration of LH, total creatine phosphokinase (CPK), and VEGF protein during reperfusion (P<0.05 vs placebo). Electron paramagnetic resonance (EPR) spectroscopy confirmed that free iron was available for oxidative catalysis in the local ischemic venous blood of supplemented patients. An increased concentration of the ascorbate radical (A.-) and alpha-phenyl-tert-butylnitrone (PBN) adducts assigned as lipid-derived alkoxyl (LO.) and alkyl (LC.) species were also detected in the peripheral blood of supplemented patients during reperfusion (P<0.05 vs ischemia). In conclusion, these findings suggest that ascorbate prophylaxis may have promoted iron-induced oxidative lipid damage via a Fenton-type reaction initiated during the ischemic phase of surgery. The subsequent release of LH into the systemic circulation may have catalyzed formation of second-generation radicals implicated in the regulation of vascular permeability and angiogenesis.

Targeting Heat Shock Proteins in Cancer: A Promising Therapeutic Approach.

PubMed

Chatterjee, Suman; Burns, Timothy F

2017-09-15

Heat shock proteins (HSPs) are a large family of chaperones that are involved in protein folding and maturation of a variety of "client" proteins protecting them from degradation, oxidative stress, hypoxia, and thermal stress. Hence, they are significant regulators of cellular proliferation, differentiation and strongly implicated in the molecular orchestration of cancer development and progression as many of their clients are well established oncoproteins in multiple tumor types. Interestingly, tumor cells are more HSP chaperonage-dependent than normal cells for proliferation and survival because the oncoproteins in cancer cells are often misfolded and require augmented chaperonage activity for correction. This led to the development of several inhibitors of HSP90 and other HSPs that have shown promise both preclinically and clinically in the treatment of cancer. In this article, we comprehensively review the roles of some of the important HSPs in cancer, and how targeting them could be efficacious, especially when traditional cancer therapies fail.

Increased microglial catalase activity in multiple sclerosis grey matter.

PubMed

Gray, Elizabeth; Kemp, Kevin; Hares, Kelly; Redondo, Julianna; Rice, Claire; Scolding, Neil; Wilkins, Alastair

2014-04-22

Chronic demyelination, on-going inflammation, axonal loss and grey matter neuronal injury are likely pathological processes that contribute to disease progression in multiple sclerosis (MS). Although the precise contribution of each process and their aetiological substrates is not fully known, recent evidence has implicated oxidative damage as a major cause of tissue injury in MS. The degree of tissue injury caused by oxidative molecules, such as reactive oxygen species (ROS), is balanced by endogenous anti-oxidant enzymes which detoxify ROS. Understanding endogenous mechanisms which protect the brain against oxidative injury in MS is important, since enhancing anti-oxidant responses is a major therapeutic strategy for preventing irreversible tissue injury in the disease. Our aims were to determine expression and activity levels of the hydrogen peroxide-reducing enzyme catalase in MS grey matter (GM). In MS GM, a catalase enzyme activity was elevated compared to control GM. We measured catalase protein expression by immune dot-blotting and catalase mRNA by a real-time polymerase chain reaction (RT-PCR). Protein analysis studies showed a strong positive correlation between catalase and microglial marker IBA-1 in MS GM. In addition, calibration of catalase mRNA level with reference to the microglial-specific transcript AIF-1 revealed an increase in this transcript in MS. This was reflected by the extent of HLA-DR immunolabeling in MS GM which was significantly elevated compared to control GM. Collectively, these observations provide evidence that microglial catalase activity is elevated in MS grey matter and may be an important endogenous anti-oxidant defence mechanism in MS. Copyright © 2014 Elsevier B.V. All rights reserved.

Oxidative stress induces vascular heme oxygenase-1 expression in ovariectomized rats.

PubMed

Lee, Yen-Mei; Cheng, Pao-Yun; Hong, Su-Fen; Chen, Shu-Ying; Lam, Kwok-Keung; Sheu, Joen-Rong; Yen, Mao-Hsiung

2005-07-01

Heme oxygenase-1 (HO-1), an inducible stress protein, has been implicated in cytoprotection against oxidative stress in vitro and in vivo. Estrogens also have antioxidant effects. This study investigated the time course of HO-1 and inducible nitric oxide synthase (iNOS) expression in the aortas of ovariectomized rats, and the regulatory relationship between the NO/NOS and the carbon monoxide/HO systems. HO-1 and iNOS protein expression was induced by ovariectomy (Ovx) and was extremely high 2-6 weeks after Ovx compared with the sham-operated group. Expression of the constitutive enzymes HO-2 and endothelial NOS did not differ significantly between sham-operated and Ovx rats. 17beta-Estradiol (E(2)) replacement reversed these changes in rats after Ovx. Long-term treatment with the antioxidant tempol significantly inhibited HO-1 and iNOS expression. The iNOS inhibitor aminoguanidine significantly suppressed the induction of HO-1. Oxidized glutathione in the hearts of Ovx rats increased gradually, with significant elevation at 3-6 weeks after Ovx compared with the sham-operated group, whereas plasma levels of NO metabolites were significantly reduced 4-6 weeks after Ovx. Treatment with the HO inhibitor zinc protoporphyrin IX blocked HO-1 induction, but significantly increased the plasma levels of NO metabolites. In conclusion, HO-1 is induced by oxidative stress resulting from E(2) depletion. The NO/iNOS system contributes to the induction of HO-1, which may subsequently suppress iNOS activity to modulate vasculoprotective effects after menopause.

Distributive Processing by the Iron(II)/α-Ketoglutarate-Dependent Catalytic Domains of the TET Enzymes Is Consistent with Epigenetic Roles for Oxidized 5-Methylcytosine Bases.

PubMed

Tamanaha, Esta; Guan, Shengxi; Marks, Katherine; Saleh, Lana

2016-08-03

The ten-eleven translocation (TET) proteins catalyze oxidation of 5-methylcytosine ((5m)C) residues in nucleic acids to 5-hydroxymethylcytosine ((5hm)C), 5-formylcytosine ((5f)C), and 5-carboxycytosine ((5ca)C). These nucleotide bases have been implicated as intermediates on the path to active demethylation, but recent reports have suggested that they might have specific regulatory roles in their own right. In this study, we present kinetic evidence showing that the catalytic domains (CDs) of TET2 and TET1 from mouse and their homologue from Naegleria gruberi, the full-length protein NgTET1, are distributive in both chemical and physical senses, as they carry out successive oxidations of a single (5m)C and multiple (5m)C residues along a polymethylated DNA substrate. We present data showing that the enzyme neither retains (5hm)C/(5f)C intermediates of preceding oxidations nor slides along a DNA substrate (without releasing it) to process an adjacent (5m)C residue. These findings contradict a recent report by Crawford et al. ( J. Am. Chem. Soc. 2016 , 138 , 730 ) claiming that oxidation of (5m)C by CD of mouse TET2 is chemically processive (iterative). We further elaborate that this distributive mechanism is maintained for TETs in two evolutionarily distant homologues and posit that this mode of function allows the introduction of (5m)C forms as epigenetic markers along the DNA.

Action of 6-amino-3-pyridinols as novel antioxidants against free radicals and oxidative stress in solution, plasma, and cultured cells.

PubMed

Omata, Yo; Saito, Yoshiro; Yoshida, Yasukazu; Jeong, Byeong-Seon; Serwa, Remigiusz; Nam, Tae-gyu; Porter, Ned A; Niki, Etsuo

2010-05-15

Free radical-mediated lipid peroxidation has been implicated in the pathogenesis of various diseases. Lipid peroxidation products are cytotoxic and they modify proteins and DNA bases, leading eventually to degenerative disorders. Various synthetic antioxidants have been developed and assessed for their capacity to inhibit lipid peroxidation and oxidative stress induced by free radicals. In this study, the capacity of novel 6-amino-2,4,5-trimethyl-3-pyridinols for scavenging peroxyl radicals, inhibiting plasma lipid peroxidation in vitro, and preventing cytotoxicity induced by glutamate, 6-hydroxydopamine, 1-methyl-4-phenylpyridium (MPP(+) ), and hydroperoxyoctadecadienoic acid was assessed. It was found that they exerted higher reactivity toward peroxyl radicals and more potent activity for inhibiting the above oxidative stress than alpha-tocopherol, the most potent natural antioxidant, except against the cytotoxicity induced by MPP(+). These results suggest that the novel 6-amino-3-pyridinols may be potent antioxidants against oxidative stress. Copyright 2010 Elsevier Inc. All rights reserved.

Repair of oxidative DNA damage by amino acids.

PubMed

Milligan, J R; Aguilera, J A; Ly, A; Tran, N Q; Hoang, O; Ward, J F

2003-11-01

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

Skeletal muscle proteomic signature and metabolic impairment in pulmonary hypertension.

PubMed

Malenfant, Simon; Potus, François; Fournier, Frédéric; Breuils-Bonnet, Sandra; Pflieger, Aude; Bourassa, Sylvie; Tremblay, Ève; Nehmé, Benjamin; Droit, Arnaud; Bonnet, Sébastien; Provencher, Steeve

2015-05-01

Exercise limitation comes from a close interaction between cardiovascular and skeletal muscle impairments. To better understand the implication of possible peripheral oxidative metabolism dysfunction, we studied the proteomic signature of skeletal muscle in pulmonary arterial hypertension (PAH). Eight idiopathic PAH patients and eight matched healthy sedentary subjects were evaluated for exercise capacity, skeletal muscle proteomic profile, metabolism, and mitochondrial function. Skeletal muscle proteins were extracted, and fractioned peptides were tagged using an iTRAQ protocol. Proteomic analyses have documented a total of 9 downregulated proteins in PAH skeletal muscles and 10 upregulated proteins compared to healthy subjects. Most of the downregulated proteins were related to mitochondrial structure and function. Focusing on skeletal muscle metabolism and mitochondrial health, PAH patients presented a decreased expression of oxidative enzymes (pyruvate dehydrogenase, p < 0.01) and an increased expression of glycolytic enzymes (lactate dehydrogenase activity, p < 0.05). These findings were supported by abnormal mitochondrial morphology on electronic microscopy, lower citrate synthase activity (p < 0.01) and lower expression of the transcription factor A of the mitochondria (p < 0.05), confirming a more glycolytic metabolism in PAH skeletal muscles. We provide evidences that impaired mitochondrial and metabolic functions found in the lungs and the right ventricle are also present in skeletal muscles of patients. • Proteomic and metabolic analysis show abnormal oxidative metabolism in PAH skeletal muscle. • EM of PAH patients reveals abnormal mitochondrial structure and distribution. • Abnormal mitochondrial health and function contribute to exercise impairments of PAH. • PAH may be considered a vascular affliction of heart and lungs with major impact on peripheral muscles.

Nitric oxide-mediated modulation of iron regulatory proteins: implication for cellular iron homeostasis.

PubMed

Kim, Sangwon; Ponka, Prem

2002-01-01

Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) that are located in the 3' untranslated region (UTR) and the 5' UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO(.), a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels and a decrease in ferritin synthesis. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels and a dramatic increase in ferritin synthesis. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels and an increase in ferritin synthesis in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO(+)-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.

Lipopolysaccharide-Induced Behavioral Alterations Are Alleviated by Sodium Phenylbutyrate via Attenuation of Oxidative Stress and Neuroinflammatory Cascade.

PubMed

Jangra, Ashok; Sriram, Chandra Shaker; Lahkar, Mangala

2016-08-01

Oxido-nitrosative stress, neuroinflammation, and reduced level of neurotrophins are implicated in the pathophysiology of anxiety and depressive illness. A few recent studies have revealed the role of endoplasmic reticulum (ER) stress in the pathophysiology of stress and depression. The aim of the present study is to investigate the neuroprotective potential of sodium phenylbutyrate (SPB), an ER stress inhibitor against lipopolysaccharide (LPS)-induced anxiety and depressive-like behavior in Swiss albino mice. Anxiety and depressive-like behavior was induced by LPS (0.83 mg/kg; i.p.) administration. Various behavioral tests were conducted to evaluate the anxiety and depressive-like behavior in mice. Real-time PCR was employed for the detection and expression of ER stress markers (78-kDa glucose-regulated protein (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)). Pretreatment with SPB significantly ameliorated the LPS-induced anxiety and depressive-like behavior as revealed by behavioral paradigm results. LPS-induced oxidative stress was ameliorated by SPB pretreatment in hippocampus (HC) and prefrontal cortex (PFC) region. Neuroinflammation was significantly reduced by SPB pretreatment in LPS-treated mice as evident from reduction in proinflammatory cytokines (IL-1β and TNF-α). Importantly, LPS administration significantly up-regulated the GRP78 mRNA expression level in the HC which suggests the involvement of unfolded protein response (UPR) in LPS-evoked behavioral anomalies. These results highlight the neuroprotective potential of SPB in LPS-induced anxiety and depressive illness model which may be partially due to inhibition of oxidative stress-neuroinflammatory cascade.

Temperature of frozen storage affects the nature and consequences of protein oxidation in beef patties.

PubMed

Utrera, Mariana; Morcuende, David; Estévez, Mario

2014-03-01

The effect of three frozen storage temperatures (-8, -18 and -80 °C) on protein oxidation in beef patties was studied through the analysis of novel oxidation markers. Additionally, the connection between lipid and protein oxidation and the impact of the latter on particular quality traits (water holding capacity, color and texture) of subsequently processed beef patties (cooking/cold-stored) were investigated. Protein oxidation was measured as the loss of tryptophan fluorescence and formation of diverse lysine oxidation products (α-aminoadipic semialdehyde, α-aminoadipic acid and Schiff bases). Lipid oxidation was assessed by levels of thiobarbituric acid reactive substances and hexanal. A significant effect of storage temperature on protein oxidation was detected. Frozen storage increased the susceptibility of meat proteins to undergo further oxidation during processing. Timely interactions were found between lipid and protein oxidation. Plausible mechanisms by which oxidative damage to proteins may have an impact in particular quality traits are thoroughly discussed. © 2013.

The Phospholipase A2 Activity of Peroxiredoxin 6.

PubMed

Fisher, Aron B

2018-05-01

Peroxiredoxin 6 (Prdx6) is a Ca2+-independent intracellular phospholipase A2 (called aiPLA2) that is localized to cytosol and acidic organelles (lysosomes and lysosomal-related organelles). Activity is minimal at cytosolic pH but is increased significantly at acidic pH, in the presence of oxidized phospholipid substrate, with protein oxidation, and with enzyme phosphorylation; maximal activity with phosphorylated aiPLA2 is ~2 μmol/min/mg protein. Prdx6 is a ″moonlighting″ protein that also expresses peroxidase and lysophosphatidylcholine acyl transferase activities.The active site for aiPLA2 activity is Ser32-H26-D140. Activity is inhibited by a serine ″protease″ inhibitor diethyl p-nitrophenyl phosphate (DENP) ,a transition state analogue 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33),and two naturally occurring proteins, surfactant protein A (SP-A) and p67phox. aiPLA2 activity has important physiological roles in the turnover (degradation and synthesis) of lung surfactant phospholipids, in the repair of peroxidized cell membranes, and in the activation of NADPH oxidase (NOX2). The enzyme has been implicated in acute lung injury, carcinogenesis, neurodegenerative diseases, diabetes, male infertility, and sundry other conditions although its specific roles have not been well defined. Protein mutations and animal models are now available to further investigate the potentially important roles of Prdx6-aiPLA2 activity in normal and pathological physiology. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Plasma Protein Oxidation and Its Correlation with Antioxidant Potential During Human Aging

PubMed Central

Pandey, Kanti Bhooshan; Mehdi, Mohd Murtaza; Maurya, Pawan Kumar; Rizvi, Syed Ibrahim

2010-01-01

Previous studies have indicated that the main molecular characteristic of aging is the progressive accumulation of oxidative damages in cellular macromolecules. Proteins are one of the main molecular targets of age-related oxidative stress, which have been observed during aging process in cellular systems. Reactive oxygen species (ROS) can lead to oxidation of amino acid side chains, formation of protein-protein cross-linkages, and oxidation of the peptide backbones. In the present study, we report the age-dependent oxidative alterations in biomarkers of plasma protein oxidation: protein carbonyls (PCO), advanced oxidation protein products (AOPPs) and plasma total thiol groups (T-SH) in the Indian population and also correlate these parameters with total plasma antioxidant potential. We show an age dependent decrease in T-SH levels and increase in PCO and AOPPs level. The alterations in the levels of these parameters correlated significantly with the total antioxidant capacity of the plasma. The levels of oxidized proteins in plasma provide an excellent biomarker of oxidative stress due to the relative long half-life of such oxidized proteins. PMID:20826915

Abiotic protein fragmentation by manganese oxide: Implications for a mechanism to supply soil biota with oligopeptides

DOE PAGES

Reardon, Patrick N.; Chacon, Stephany S.; Walter, Eric D.; ...

2016-03-14

Proteins facilitate a wide range of chemical transformations important in soil as well as being a major reservoir of soil nitrogen themselves. The interactions and reactions of proteins with soils and minerals are of key importance to our understanding of their functional persistence in the environment. We combined NMR and EPR spectroscopies to distinguish the reaction of a model protein with a redox active mineral surface (Birnessite, MnO 2) from its response to a redox neutral phyllosilicate (Kaolinite). Our data demonstrate that birnessite fragments the model protein while kaolinite has little impact on the protein structure. NMR and EPR spectroscopiesmore » are shown to be valuable tools to observe these reactions and capture the extent of protein transformation together with the extent of mineral response. These data suggest that mineral surfaces can have both promoting and retarding roles in terrestrial nitrogen cycling, with redox active minerals acting as accelerators by catalyzing the breakdown of proteins and proteinaceous materials while phyllosilicates are more likely to act as preservative media.« less

Nuclear localization of human spermine oxidase isoforms – possible implications in drug response and disease etiology

PubMed Central

Murray-Stewart, Tracy; Wang, Yanlin; Goodwin, Andrew; Hacker, Amy; Meeker, Alan; Casero, Robert A.

2013-01-01

The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO-produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase-1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI-H157 human non-small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation-induced carcinogenesis. PMID:18422650

Silibinin Attenuates Sulfur Mustard Analog-Induced Skin Injury by Targeting Multiple Pathways Connecting Oxidative Stress and Inflammation

PubMed Central

Tewari-Singh, Neera; Jain, Anil K.; Inturi, Swetha; Agarwal, Chapla; White, Carl W.; Agarwal, Rajesh

2012-01-01

Chemical warfare agent sulfur mustard (HD) inflicts delayed blistering and incapacitating skin injuries. To identify effective countermeasures against HD-induced skin injuries, efficacy studies were carried out employing HD analog 2-chloroethyl ethyl sulfide (CEES)-induced injury biomarkers in skin cells and SKH-1 hairless mouse skin. The data demonstrate strong therapeutic efficacy of silibinin, a natural flavanone, in attenuating CEES-induced skin injury and oxidative stress. In skin cells, silibinin (10 µM) treatment 30 min after 0.35/0.5 mM CEES exposure caused a significant (p<0.05) reversal in CEES-induced decrease in cell viability, apoptotic and necrotic cell death, DNA damage, and an increase in oxidative stress. Silibinin (1 mg) applied topically to mouse skin 30 min post-CEES exposure (2 mg), was effective in reversing CEES-induced increases in skin bi-fold (62%) and epidermal thickness (85%), apoptotic cell death (70%), myeloperoxidase activity (complete reversal), induction of iNOS, COX-2, and MMP-9 protein levels (>90%), and activation of transcription factors NF-κB and AP-1 (complete reversal). Similarly, silibinin treatment was also effective in attenuating CEES-induced oxidative stress measured by 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrolline N-oxide protein adduct formation, and 8-oxo-2-deoxyguanosine levels. Since our previous studies implicated oxidative stress, in part, in CEES-induced toxic responses, the reversal of CEES-induced oxidative stress and other toxic effects by silibinin in this study indicate its pleiotropic therapeutic efficacy. Together, these findings support further optimization of silibinin in HD skin toxicity model to develop a novel effective therapy for skin injuries by vesicants. PMID:23029417

Brain-targeted ACE2 overexpression attenuates neurogenic hypertension by inhibiting COX mediated inflammation

PubMed Central

Sriramula, Srinivas; Xia, Huijing; Xu, Ping; Lazartigues, Eric

2014-01-01

Overactivity of the renin angiotensin system (RAS), oxidative stress, and cyclooxygenases (COX) in the brain are implicated in the pathogenesis of hypertension. We previously reported that Angiotensin-Converting Enzyme 2 (ACE2) overexpression in the brain attenuates the development of DOCA-salt hypertension, a neurogenic hypertension model with enhanced brain RAS and sympathetic activity. To elucidate the mechanisms involved, we investigated whether oxidative stress, mitogen activated protein kinase signaling and cyclooxygenase (COX) activation in the brain are modulated by ACE2 in neurogenic hypertension. DOCA-salt hypertension significantly increased expression of Nox-2 (+61 ±5 %), Nox-4 (+50 ±13 %) and nitrotyrosine (+89 ±32 %) and reduced activity of the antioxidant enzymes, catalase (−29 ±4 %) and SOD (−31 ±7 %), indicating increased oxidative stress in the brain of non-transgenic mice. This increased oxidative stress was attenuated in transgenic mice overexpressing ACE2 in the brain. DOCA-salt-induced reduction of nNOS expression (−26 ±7 %) and phosphorylated eNOS/total eNOS (−30 ±3 %), and enhanced phosphorylation of Akt and ERK1/2 in the paraventricular nucleus (PVN), were reversed by ACE2 overexpression. In addition, ACE2 overexpression blunted the hypertension-mediated increase in gene and protein expression of COX-1 and COX-2 in the PVN. Furthermore, gene silencing of either COX-1 or COX-2 in the brain, reduced microglial activation and accompanied neuro-inflammation, ultimately attenuating DOCA-salt hypertension. Together, these data provide evidence that brain ACE2 overexpression reduces oxidative stress and COX-mediated neuro-inflammation, improves anti-oxidant and nitric oxide signaling, and thereby attenuates the development of neurogenic hypertension. PMID:25489058

Silibinin attenuates sulfur mustard analog-induced skin injury by targeting multiple pathways connecting oxidative stress and inflammation.

PubMed

Tewari-Singh, Neera; Jain, Anil K; Inturi, Swetha; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

2012-01-01

Chemical warfare agent sulfur mustard (HD) inflicts delayed blistering and incapacitating skin injuries. To identify effective countermeasures against HD-induced skin injuries, efficacy studies were carried out employing HD analog 2-chloroethyl ethyl sulfide (CEES)-induced injury biomarkers in skin cells and SKH-1 hairless mouse skin. The data demonstrate strong therapeutic efficacy of silibinin, a natural flavanone, in attenuating CEES-induced skin injury and oxidative stress. In skin cells, silibinin (10 µM) treatment 30 min after 0.35/0.5 mM CEES exposure caused a significant (p<0.05) reversal in CEES-induced decrease in cell viability, apoptotic and necrotic cell death, DNA damage, and an increase in oxidative stress. Silibinin (1 mg) applied topically to mouse skin 30 min post-CEES exposure (2 mg), was effective in reversing CEES-induced increases in skin bi-fold (62%) and epidermal thickness (85%), apoptotic cell death (70%), myeloperoxidase activity (complete reversal), induction of iNOS, COX-2, and MMP-9 protein levels (>90%), and activation of transcription factors NF-κB and AP-1 (complete reversal). Similarly, silibinin treatment was also effective in attenuating CEES-induced oxidative stress measured by 4-hydroxynonenal and 5,5-dimethyl-2-(8-octanoic acid)-1-pyrolline N-oxide protein adduct formation, and 8-oxo-2-deoxyguanosine levels. Since our previous studies implicated oxidative stress, in part, in CEES-induced toxic responses, the reversal of CEES-induced oxidative stress and other toxic effects by silibinin in this study indicate its pleiotropic therapeutic efficacy. Together, these findings support further optimization of silibinin in HD skin toxicity model to develop a novel effective therapy for skin injuries by vesicants.

The Effect of Neighboring Methionine Residue on Tyrosine Nitration & Oxidation in Peptides Treated with MPO, H2O2, & NO2- or Peroxynitrite and Bicarbonate: Role of Intramolecular Electron-Transfer Mechanism?

PubMed Central

Zhang, Hao; Zielonka, Jacek; Sikora, Adam; Joseph, Joy; Xu, Yingkai; Kalyanaraman, B.

2009-01-01

Recent reports suggest that intramolecular electron-transfer reactions can profoundly affect the site and specificity of tyrosyl nitration and oxidation in peptides and proteins. Here we investigated the effects of methionine on tyrosyl nitration and oxidation induced by myeloperoxidase (MPO), H2O2 and NO2- and peroxynitrite (ONOO-) or ONOO- and bicarbonate (HCO3-) in model peptides, tyrosylmethionine (YM), tyrosylphenylalanine (YF) and tyrosine. Nitration and oxidation products of these peptides were analysed by HPLC with UV/Vis and fluorescence detection, and mass spectrometry; radical intermediates were identified by electron paramagnetic resonance (EPR)-spin-trapping. We have previously shown (Zhang et al., J. Biol. Chem. (2005) 280, 40684-40698) that oxidation and nitration of tyrosyl residue was inhibited in tyrosylcysteine(YC)-type peptides as compared to free tyrosine. Here we show that methionine, another sulfur-containing amino acid, does not inhibit nitration and oxidation of a neighboring tyrosine residue in the presence of ONOO- (or ONOOCO2-) or MPO/H2O2/NO2- system. Nitration of tyrosyl residue in YM was actually stimulated under the conditions of in situ generation of ONOO- (formed by reaction of superoxide with nitric oxide during SIN-1 decomposition), as compared to YF, YC and tyrosine. The dramatic variations in tyrosyl nitration profiles caused by methionine and cysteine residues have been attributed to differences in the direction of intramolecular electron transfer mechanism in these peptides. Further confirmation of HPLC data analysis was obtained by steady-state radiolysis and photolysis experiments. Potential implications of the intramolecular electron-transfer mechanism in mediating selective nitration of protein tyrosyl groups are discussed. PMID:19056332

Proteomic Analyses of Cysteine Redox in High-Fat-Fed and Fasted Mouse Livers: Implications for Liver Metabolic Homeostasis.

PubMed

Li, Yixing; Luo, Zupeng; Wu, Xilong; Zhu, Jun; Yu, Kai; Jin, Yi; Zhang, Zhiwang; Zhao, Shuhong; Zhou, Lei

2018-01-05

Intensive oxidative stress occurs during high-fat-diet-induced hepatic fat deposition, suggesting a critical role for redox signaling in liver metabolism. Intriguingly, evidence shows that fasting could also result in redox-profile changes largely through reduced oxidant or increased antioxidant levels. However, a comprehensive landscape of redox-modified hepatic substrates is lacking, thereby hindering our understanding of liver metabolic homeostasis. We employed a proteomic approach combining iodoacetyl tandem mass tag and nanoliquid chromatography tandem mass spectrometry to quantitatively probe the effects of high-fat feeding and fasting on in vivo redox-based cysteine modifications. Compared with control groups, ∼60% of cysteine residues exhibited downregulated oxidation ratios by fasting, whereas ∼94% of these ratios were upregulated by high-fat feeding. Importantly, in fasted livers, proteins exhibiting diminished cysteine oxidation were annotated in pathways associated with fatty acid metabolism, carbohydrate metabolism, insulin, peroxisome proliferator-activated receptors, and oxidative respiratory chain signaling, suggesting that fasting-induced redox changes targeted major metabolic pathways and consequently resulted in hepatic lipid accumulation.

Dysfunction of pulmonary surfactant mediated by phospholipid oxidation is cholesterol-dependent.

PubMed

Al-Saiedy, Mustafa; Pratt, Ryan; Lai, Patrick; Kerek, Evan; Joyce, Heidi; Prenner, Elmar; Green, Francis; Ling, Chang-Chun; Veldhuizen, Ruud; Ghandorah, Salim; Amrein, Matthias

2018-04-01

Pulmonary surfactant forms a cohesive film at the alveolar air-lung interface, lowering surface tension, and thus reducing the work of breathing and preventing atelectasis. Surfactant function becomes impaired during inflammation due to degradation of the surfactant lipids and proteins by free radicals. In this study, we examine the role of reactive nitrogen (RNS) and oxygen (ROS) species on surfactant function with and without physiological cholesterol levels (5-10%). Surface activity was assessed in vitro in a captive bubble surfactometer (CBS). Surfactant chemistry, monolayer fluidity and thermodynamic behavior were also recorded before and after oxidation. We report that physiologic amounts of cholesterol combined with oxidation results in severe impairment of surfactant function. We also show that surfactant polyunsaturated phospholipids are the most susceptible to oxidative alteration. Membrane thermodynamic experiments showed significant surfactant film stiffening after free radical exposure in the presence of cholesterol. These results point to a previously unappreciated role for cholesterol in amplifying defects in surface activity caused by oxidation of pulmonary surfactant, a finding that may have implications for treating several lung diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Marine Cyanobacteria Compounds with Anticancer Properties: A Review on the Implication of Apoptosis

PubMed Central

Costa, Margarida; Costa-Rodrigues, João; Fernandes, Maria Helena; Barros, Piedade; Vasconcelos, Vitor; Martins, Rosário

2012-01-01

Marine cyanobacteria have been considered a rich source of secondary metabolites with potential biotechnological applications, namely in the pharmacological field. Chemically diverse compounds were found to induce cytoxicity, anti-inflammatory and antibacterial activities. The potential of marine cyanobacteria as anticancer agents has however been the most explored and, besides cytotoxicity in tumor cell lines, several compounds have emerged as templates for the development of new anticancer drugs. The mechanisms implicated in the cytotoxicity of marine cyanobacteria compounds in tumor cell lines are still largely overlooked but several studies point to an implication in apoptosis. This association has been related to several apoptotic indicators such as cell cycle arrest, mitochondrial dysfunctions and oxidative damage, alterations in caspase cascade, alterations in specific proteins levels and alterations in the membrane sodium dynamics. In the present paper a compilation of the described marine cyanobacterial compounds with potential anticancer properties is presented and a review on the implication of apoptosis as the mechanism of cell death is discussed. PMID:23170077

Hypochlorous and peracetic acid induced oxidation of dairy proteins.

PubMed

Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

2011-02-09

Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

Reactive metabolites and antioxidant gene polymorphisms in Type 2 diabetes mellitus☆

PubMed Central

Banerjee, Monisha; Vats, Pushpank

2013-01-01

Type 2 diabetes mellitus (T2DM), by definition is a heterogeneous, multifactorial, polygenic syndrome which results from insulin receptor dysfunction. It is an outcome of oxidative stress caused by interactions of reactive metabolites (RMs) interactions with lipids, proteins and other mechanisms of human body. Production of RMs mainly superoxide (O2−) has been found in a variety of predominating cellular enzyme systems including NAD(P)H oxidase, xanthine oxidase (XO), cyclooxygenase (COX), uncoupled endothelial nitric oxide synthase (eNOS) and myeloperoxidase (MPO). The four main RM related molecular mechanisms are: increased polyol pathway flux; increased advanced glycation end-product (AGE) formation; activation of protein kinase C (PKC) isoforms and increased hexosamine pathway flux which have been implicated in glucose-mediated vascular damage. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), nitric oxide synthase (NOS) are antioxidant enzymes involved in scavenging RMs in normal individuals. Functional polymorphisms of these antioxidant enzymes have been reported to be involved in pathogenesis of T2DM individuals. The low levels of antioxidant enzymes or their non-functionality results in excessive RMs which initiate stress related pathways thereby leading to insulin resistance and T2DM. An attempt has been made to review the role of RMs and antioxidant enzymes in oxidative stress resulting in T2DM. PMID:25460725

Whole genome siRNA cell-based screen links mitochondria to Akt signaling network through uncoupling of electron transport chain

PubMed Central

Senapedis, William T.; Kennedy, Caleb J.; Boyle, Patrick M.; Silver, Pamela A.

2011-01-01

Forkhead transcription factors (FOXOs) alter a diverse array of cellular processes including the cell cycle, oxidative stress resistance, and aging. Insulin/Akt activation directs phosphorylation and cytoplasmic sequestration of FOXO away from its target genes and serves as an endpoint of a complex signaling network. Using a human genome small interfering RNA (siRNA) library in a cell-based assay, we identified an extensive network of proteins involved in nuclear export, focal adhesion, and mitochondrial respiration not previously implicated in FOXO localization. Furthermore, a detailed examination of mitochondrial factors revealed that loss of uncoupling protein 5 (UCP5) modifies the energy balance and increases free radicals through up-regulation of uncoupling protein 3 (UCP3). The increased superoxide content induces c-Jun N-terminal kinase 1 (JNK1) kinase activity, which in turn affects FOXO localization through a compensatory dephosphorylation of Akt. The resulting nuclear FOXO increases expression of target genes, including mitochondrial superoxide dismutase. By connecting free radical defense and mitochondrial uncoupling to Akt/FOXO signaling, these results have implications in obesity and type 2 diabetes development and the potential for therapeutic intervention. PMID:21460183

Whole genome siRNA cell-based screen links mitochondria to Akt signaling network through uncoupling of electron transport chain.

PubMed

Senapedis, William T; Kennedy, Caleb J; Boyle, Patrick M; Silver, Pamela A

2011-05-15

Forkhead transcription factors (FOXOs) alter a diverse array of cellular processes including the cell cycle, oxidative stress resistance, and aging. Insulin/Akt activation directs phosphorylation and cytoplasmic sequestration of FOXO away from its target genes and serves as an endpoint of a complex signaling network. Using a human genome small interfering RNA (siRNA) library in a cell-based assay, we identified an extensive network of proteins involved in nuclear export, focal adhesion, and mitochondrial respiration not previously implicated in FOXO localization. Furthermore, a detailed examination of mitochondrial factors revealed that loss of uncoupling protein 5 (UCP5) modifies the energy balance and increases free radicals through up-regulation of uncoupling protein 3 (UCP3). The increased superoxide content induces c-Jun N-terminal kinase 1 (JNK1) kinase activity, which in turn affects FOXO localization through a compensatory dephosphorylation of Akt. The resulting nuclear FOXO increases expression of target genes, including mitochondrial superoxide dismutase. By connecting free radical defense and mitochondrial uncoupling to Akt/FOXO signaling, these results have implications in obesity and type 2 diabetes development and the potential for therapeutic intervention.

Oxidative stress and protein aggregation during biological aging.

PubMed

Squier, T C

2001-09-01

Biological aging is a fundamental process that represents the major risk factor with respect to the development of cancer, neurodegenerative, and cardiovascular diseases in vertebrates. It is, therefore, evident that the molecular mechanisms of aging are fundamental to understand many disease processes. In this regard, the oxidation and nitration of intracellular proteins and the formation of protein aggregates have been suggested to underlie the loss of cellular function and the reduced ability of senescent animals to withstand physiological stresses. Since oxidatively modified proteins are thermodynamically unstable and assume partially unfolded tertiary structures that readily form aggregates, it is likely that oxidized proteins are intermediates in the formation of amyloid fibrils. It is, therefore, of interest to identify oxidatively sensitive protein targets that may play a protective role through their ability to down-regulate energy metabolism and the consequent generation of reactive oxygen species (ROS). In this respect, the maintenance of cellular calcium gradients represents a major energetic expense, which links alterations in intracellular calcium levels to ATP utilization and the associated generation of ROS through respiratory control mechanisms. The selective oxidation or nitration of the calcium regulatory proteins calmodulin and Ca-ATPase that occurs in vivo during aging and under conditions of oxidative stress may represent an adaptive response to oxidative stress that functions to down-regulate energy metabolism and the associated generation of ROS. Since these calcium regulatory proteins are also preferentially oxidized or nitrated under in vitro conditions, these results suggest an enhanced sensitivity of these critical calcium regulatory proteins, which modulate signal transduction processes and intracellular energy metabolism, to conditions of oxidative stress. Thus, the selective oxidation of critical signal transduction proteins probably represents a regulatory mechanism that functions to minimize the generation of ROS through respiratory control mechanisms. The reduction of the rate of ROS generation, in turn, will promote cellular survival under conditions of oxidative stress, when reactive oxygen and nitrogen species overwhelm cellular antioxidant defense systems, by minimizing the non-selective oxidation of a range of biomolecules. Since protein aggregation occurs if protein repair and degradative systems are unable to act upon oxidized proteins and restore cellular function, the reduction of the oxidative load on the cell by the down-regulation of the electron transport chain functions to minimize protein aggregation. Thus, ROS function as signaling molecules that fine-tune cellular metabolism through the selective oxidation or nitration of calcium regulatory proteins in order to minimize wide-spread oxidative damage and protein aggregation. Oxidative damage to cellular proteins, the loss of calcium homeostasis and protein aggregation contribute to the formation of amyloid deposits that accumulate during biological aging. Critical to understand the relationship between these processes and biological aging is the identification of oxidatively sensitive proteins that modulate energy utilization and the associated generation of ROS. In this latter respect, oxidative modifications to the calcium regulatory proteins calmodulin (CaM) and the sarco/endoplasmic reticulum Ca-ATPase (SERCA) function to down-regulate ATP utilization and the associated generation of ROS associated with replenishing intracellular ATP through oxidative phosphorylation. Reductions in the rate of ROS generation, in turn, will minimize protein oxidation and facilitate intracellular repair and degradative systems that function to eliminate damaged and partially unfolded proteins. Since the rates of protein repair or degradation compete with the rate of protein aggregation, the modulation of intracellular calcium concentrations and energy metabolism through the selective oxidation or nitration of critical signal transduction proteins (i.e. CaM or SERCA) is thought to maintain cellular function by minimizing protein aggregation and amyloid formation. Age-dependent increases in the rate of ROS generation or declines in cellular repair or degradation mechanisms will increase the oxidative load on the cell, resulting in corresponding increases in the concentrations of oxidized proteins and the associated formation of amyloid.

Complete genome of Nitrosospira briensis C-128, an ammonia-oxidizing bacterium from agricultural soil

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rice, Marlen C.; Norton, Jeanette M.; Valois, Frederica

Nitrosospira briensis C-128 is an ammonia-oxidizing bacterium isolated from an acid agricultural soil. N. briensis C-128 was sequenced with PacBio RS technologies at the DOE-Joint Genome Institute through their Community Science Program (2010). The high-quality finished genome contains one chromosome of 3.21 Mb and no plasmids. We identified 3073 gene models, 3018 of which are protein coding. The two-way average nucleotide identity between the chromosomes of Nitrosospira multiformis ATCC 25196 and Nitrosospira briensis C-128 was found to be 77.2 %. Multiple copies of modules encoding chemolithotrophic metabolism were identified in their genomic context. The gene inventory supports chemolithotrophic metabolism withmore » implications for function in soil environments.« less

Complete genome of Nitrosospira briensis C-128, an ammonia-oxidizing bacterium from agricultural soil

DOE PAGES

Rice, Marlen C.; Norton, Jeanette M.; Valois, Frederica; ...

2016-07-28

Nitrosospira briensis C-128 is an ammonia-oxidizing bacterium isolated from an acid agricultural soil. N. briensis C-128 was sequenced with PacBio RS technologies at the DOE-Joint Genome Institute through their Community Science Program (2010). The high-quality finished genome contains one chromosome of 3.21 Mb and no plasmids. We identified 3073 gene models, 3018 of which are protein coding. The two-way average nucleotide identity between the chromosomes of Nitrosospira multiformis ATCC 25196 and Nitrosospira briensis C-128 was found to be 77.2 %. Multiple copies of modules encoding chemolithotrophic metabolism were identified in their genomic context. The gene inventory supports chemolithotrophic metabolism withmore » implications for function in soil environments.« less

Protein Oxidation Implicated as the Primary Determinant of Bacterial Radioresistance

DTIC Science & Technology

2007-04-01

the facultative anaerobic, radio- resistant bacterium Lactobacillus plantarum [1,22] accumulates 20–25 mM Mn(II) [23]. For an irradiated cell...in dH2O at 0 8C and pre-conditioned to be anaerobic, H2O2 was released by D. radiodurans (;23 105 M) and L. plantarum (;6 3 105 M), in which the...non-irradiated D. radiodurans or L. plantarum control samples, nor by irradiated S. oneidensis (Figure 5A), which accumulates substantially more Fe

ALTERATIONS OF MACROPHAGE FUNCTIONS BY MEDIATORS FROM LYMPHOCYTES

PubMed Central

Nathan, Carl F.; Karnovsky, Manfred L.; David, John R.

1971-01-01

Sensitized lymphocytes were incubated in vitro with the specific antigen Supernatants from these cultures were chromatographed on Sephadex G-100 columns. Supernatant fractions containing MIF, chemotactic factor, and lymphotoxin, but free of antigen and antibody, were incubated with normal peritoneal exudate macrophages. Macrophage adherence, phagocytosis, spreading, motility, and direct hexose monophosphate oxidation were enhanced, while protein synthesis was unaffected. Thus, antigen-stimulated lymphocytes secrete a factor or factors which enhance certain macrophage functions. Implications for models of cellular immunity and cellular hypersensitivity are discussed. PMID:5576335

Redox Proteomics: A Key Tool for New Insights into Protein Modification with Relevance to Disease.

PubMed

Butterfield, D Allan; Perluigi, Marzia

2017-03-01

Oxidatively modified proteins are characterized by elevations in protein-resident carbonyls or 3-nitrotyrosine, measures of protein oxidation, or protein bound reactive alkenals such as 4-hydroxy-2-nonenal, a measure of lipid peroxidation. Oxidatively modified proteins nearly always have altered structure and function. Redox proteomics is that branch of proteomics used to identify oxidized proteins and determine the extent and location of oxidative modifications in the proteomes of interest. This technique nearly always employs mass spectrometry as the major platform to achieve the goals of identifying the target proteins. Once identified, oxidatively modified proteins can be placed in specific molecular pathways to provide insights into protein oxidation and human disease. Both original research and review articles are included in this Forum on Redox Proteomics. The topics related to redox proteomics range from basic chemistry of sulfur radical-induced redox modifications in proteins, to the thiol secretome and inflammatory network, to reversible thiol oxidation in proteomes, to the role of glutamine synthetase in peripheral and central environments on inflammation and insulin resistance, to bioanalytical aspects of tyrosine nitrated proteins, to protein oxidation in human smokers and models thereof, and to Alzheimer disease, including articles on the brain ubiquitinylome and the "triangle of death" composed of oxidatively modified proteins involved in energy metabolism, mammalian target of rampamycin activation, and the proteostasis network. This Forum on Redox Proteomics is both timely and a critically important resource to highlight one of the key tools needed to better understand protein structure and function in oxidative environments in health and disease. Antioxid. Redox Signal. 26, 277-279.

Nickel Inhibits Mitochondrial Fatty Acid Oxidation

PubMed Central

Uppala, Radha; McKinney, Richard W.; Brant, Kelly A.; Fabisiak, James P.; Goetzman, Eric S.

2015-01-01

Nickel exposure is associated with changes in cellular energy metabolism which may contribute to its carcinogenic properties. Here, we demonstrate that nickel strongly represses mitochondrial fatty acid oxidation—the pathway by which fatty acids are catabolized for energy—in both primary human lung fibroblasts and mouse embryonic fibroblasts. At the concentrations used, nickel suppresses fatty acid oxidation without globally suppressing mitochondrial function as evidenced by increased glucose oxidation to CO2. Pre-treatment with L-carnitine, previously shown to prevent nickel-induced mitochondrial dysfunction in neuroblastoma cells, did not prevent the inhibition of fatty acid oxidation. The effect of nickel on fatty acid oxidation occurred only with prolonged exposure (>5 hr), suggesting that direct inhibition of the active sites of metabolic enzymes is not the mechanism of action. Nickel is a known hypoxia-mimetic that activates hypoxia inducible factor-1α (HIF1α). Nickel-induced inhibition of fatty acid oxidation was blunted in HIF1α knockout fibroblasts, implicating HIF1α as one contributor to the mechanism. Additionally, nickel down-regulated the protein levels of the key fatty acid oxidation enzyme very long-chain acyl-CoA dehydrogenase (VLCAD) in a dose-dependent fashion. In conclusion, inhibition of fatty acid oxidation by nickel, concurrent with increased glucose metabolism, represents a form of metabolic reprogramming that may contribute to nickel-induced carcinogenesis. PMID:26051273

Kidney-targeted inhibition of protein kinase C-α ameliorates nephrotoxic nephritis with restoration of mitochondrial dysfunction.

PubMed

Kvirkvelia, Nino; McMenamin, Malgorzata; Warren, Marie; Jadeja, Ravirajsinh N; Kodeboyina, Sai Karthik; Sharma, Ashok; Zhi, Wenbo; O'Connor, Paul M; Raju, Raghavan; Lucas, Rudolf; Madaio, Michael P

2018-05-04

To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis.

PubMed

Tampo, Yoshiko; Kotamraju, Srigiridhar; Chitambar, Christopher R; Kalivendi, Shasi V; Keszler, Agnes; Joseph, Joy; Kalyanaraman, B

2003-01-10

Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H2O2-dependent intracellular oxidation of DCFH to a green fluorescent product, 2',7'-dichlorofluorescein (DCF), required the uptake of extracellular iron transported through a transferrin receptor (TfR) in endothelial cells. H2O2-induced DCF fluorescence was inhibited by the monoclonal IgA-class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H2O2-mediated inactivation of cytosolic aconitase was responsible for activation of iron regulatory protein-1 and increased expression of TfR, resulting in an increased iron uptake into endothelial cells. H2O2-mediated caspase-3 proteolytic activation was inhibited by anti-TfR antibody. Similar results were obtained in the presence of a lipid hydroperoxide. We conclude that hydroperoxide-induced DCFH oxidation and endothelial cell apoptosis required the uptake of extracellular iron by the TfR-dependent iron transport mechanism and that the peroxide-induced iron signaling, in general, has broader implications in oxidative vascular biology.

Effects of solution conditions on methionine oxidation in albinterferon alfa-2b and the role of oxidation in its conformation and aggregation.

PubMed

Chou, Danny K; Krishnamurthy, Rajesh; Manning, Mark Cornell; Randolph, Theodore W; Carpenter, John F

2013-02-01

Physical and chemical degradation of therapeutic proteins can occur simultaneously. In this study, our first objective was to investigate how solution conditions that impact conformational stability of albinterferon alfa-2b, a recombinant fusion protein, modulate rates of methionine (Met) oxidation. Another objective of this work was to determine whether oxidation affects conformation and rate of aggregation of the protein. The protein was subjected to oxidation in solutions of varying pH, ionic strength, and excipients by the addition of 0.02% tertiary-butyl hydroperoxide (TBHP). The rate of formation of Met-sulfoxide species was monitored by reversed-phase high-performance liquid chromatography and compared across solution conditions. Albinterferon alfa-2b exhibited susceptibility to Met oxidation during exposure to TBHP that was highly dependent on solution parameters, but there was not a clear correlation between oxidation rate and protein conformational stability. Met oxidation resulted in significant perturbation of both secondary and tertiary structure of albinterferon alfa-2b as shown by both far-ultraviolet (UV) and near-UV circular dichroism. Moreover, oxidation of the protein caused a noticeable reduction in the protein's resistance to thermal denaturation. Surprisingly, despite its negative effect on solution structure and conformational stability, oxidation actually reduced the protein's aggregation rate during agitation at room temperature as well as during quiescent incubation at 40°C. Oxidation of the protein resulted in improved colloidal stability of the protein, which is manifested by a more positive B(22) value in the oxidized protein. Thus, the reduced aggregation rate after oxidation suggests that increased colloidal stability of oxidized albinterferon alfa-2b counteracted oxidation-induced decreases in conformational stability. Copyright © 2012 Wiley Periodicals, Inc.

Positive Darwinian Selection in the Piston That Powers Proton Pumps in Complex I of the Mitochondria of Pacific Salmon

PubMed Central

Garvin, Michael R.; Bielawski, Joseph P.; Gharrett, Anthony J.

2011-01-01

The mechanism of oxidative phosphorylation is well understood, but evolution of the proteins involved is not. We combined phylogenetic, genomic, and structural biology analyses to examine the evolution of twelve mitochondrial encoded proteins of closely related, yet phenotypically diverse, Pacific salmon. Two separate analyses identified the same seven positively selected sites in ND5. A strong signal was also detected at three sites of ND2. An energetic coupling analysis revealed several structures in the ND5 protein that may have co-evolved with the selected sites. These data implicate Complex I, specifically the piston arm of ND5 where it connects the proton pumps, as important in the evolution of Pacific salmon. Lastly, the lineage to Chinook experienced rapid evolution at the piston arm. PMID:21969854

Positive Darwinian selection in the piston that powers proton pumps in complex I of the mitochondria of Pacific salmon.

PubMed

Garvin, Michael R; Bielawski, Joseph P; Gharrett, Anthony J

2011-01-01

The mechanism of oxidative phosphorylation is well understood, but evolution of the proteins involved is not. We combined phylogenetic, genomic, and structural biology analyses to examine the evolution of twelve mitochondrial encoded proteins of closely related, yet phenotypically diverse, Pacific salmon. Two separate analyses identified the same seven positively selected sites in ND5. A strong signal was also detected at three sites of ND2. An energetic coupling analysis revealed several structures in the ND5 protein that may have co-evolved with the selected sites. These data implicate Complex I, specifically the piston arm of ND5 where it connects the proton pumps, as important in the evolution of Pacific salmon. Lastly, the lineage to Chinook experienced rapid evolution at the piston arm.

Selective Sensitization of Zinc Finger Protein Oxidation by Reactive Oxygen Species through Arsenic Binding*

PubMed Central

Zhou, Xixi; Cooper, Karen L.; Sun, Xi; Liu, Ke J.; Hudson, Laurie G.

2015-01-01

Cysteine oxidation induced by reactive oxygen species (ROS) on redox-sensitive targets such as zinc finger proteins plays a critical role in redox signaling and subsequent biological outcomes. We found that arsenic exposure led to oxidation of certain zinc finger proteins based on arsenic interaction with zinc finger motifs. Analysis of zinc finger proteins isolated from arsenic-exposed cells and zinc finger peptides by mass spectrometry demonstrated preferential oxidation of C3H1 and C4 zinc finger configurations. C2H2 zinc finger proteins that do not bind arsenic were not oxidized by arsenic-generated ROS in the cellular environment. The findings suggest that selectivity in arsenic binding to zinc fingers with three or more cysteines defines the target proteins for oxidation by ROS. This represents a novel mechanism of selective protein oxidation and demonstrates how an environmental factor may sensitize certain target proteins for oxidation, thus altering the oxidation profile and redox regulation. PMID:26063799

Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres.

PubMed

Lin, Jiandie; Wu, Hai; Tarr, Paul T; Zhang, Chen-Yu; Wu, Zhidan; Boss, Olivier; Michael, Laura F; Puigserver, Pere; Isotani, Eiji; Olson, Eric N; Lowell, Bradford B; Bassel-Duby, Rhonda; Spiegelman, Bruce M

2002-08-15

The biochemical basis for the regulation of fibre-type determination in skeletal muscle is not well understood. In addition to the expression of particular myofibrillar proteins, type I (slow-twitch) fibres are much higher in mitochondrial content and are more dependent on oxidative metabolism than type II (fast-twitch) fibres. We have previously identified a transcriptional co-activator, peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1 alpha), which is expressed in several tissues including brown fat and skeletal muscle, and that activates mitochondrial biogenesis and oxidative metabolism. We show here that PGC-1 alpha is expressed preferentially in muscle enriched in type I fibres. When PGC-1 alpha is expressed at physiological levels in transgenic mice driven by a muscle creatine kinase (MCK) promoter, a fibre type conversion is observed: muscles normally rich in type II fibres are redder and activate genes of mitochondrial oxidative metabolism. Notably, putative type II muscles from PGC-1 alpha transgenic mice also express proteins characteristic of type I fibres, such as troponin I (slow) and myoglobin, and show a much greater resistance to electrically stimulated fatigue. Using fibre-type-specific promoters, we show in cultured muscle cells that PGC-1 alpha activates transcription in cooperation with Mef2 proteins and serves as a target for calcineurin signalling, which has been implicated in slow fibre gene expression. These data indicate that PGC-1 alpha is a principal factor regulating muscle fibre type determination.

PKC1 is essential for protection against both oxidative and nitrosative stresses, cell integrity, and normal manifestation of virulence factors in the pathogenic fungus Cryptococcus neoformans.

PubMed

Gerik, Kimberly J; Bhimireddy, Sujit R; Ryerse, Jan S; Specht, Charles A; Lodge, Jennifer K

2008-10-01

Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a "top-down" approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Delta strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.

PKC1 Is Essential for Protection against both Oxidative and Nitrosative Stresses, Cell Integrity, and Normal Manifestation of Virulence Factors in the Pathogenic Fungus Cryptococcus neoformans▿ †

PubMed Central

Gerik, Kimberly J.; Bhimireddy, Sujit R.; Ryerse, Jan S.; Specht, Charles A.; Lodge, Jennifer K.

2008-01-01

Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a “top-down” approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Δ strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism. PMID:18689526

Protein Kinase-C Beta Contributes to Impaired Endothelial Insulin Signaling in Humans with Diabetes Mellitus

PubMed Central

Tabit, Corey E; Shenouda, Sherene M; Holbrook, Monica; Fetterman, Jessica L; Kiani, Soroosh; Frame, Alissa A; Kluge, Matthew A; Held, Aaron; Dohadwala, Mustali; Gokce, Noyan; Farb, Melissa; Rosenzweig, James; Ruderman, Neil; Vita, Joseph A; Hamburg, Naomi M

2013-01-01

Background Abnormal endothelial function promotes atherosclerotic vascular disease in diabetes. Experimental studies indicate that disruption of endothelial insulin signaling through the activity of protein kinase C-β (PKCβ) and nuclear factor κB (NFκB) reduces nitric oxide availability. We sought to establish whether similar mechanisms operate in the endothelium in human diabetes mellitus. Methods and Results We measured protein expression and insulin response in freshly isolated endothelial cells from patients with Type 2 diabetes mellitus (n=40) and non-diabetic controls (n=36). Unexpectedly, we observed 1.7-fold higher basal endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 in patients with diabetes (P=0.007) without a difference in total eNOS expression. Insulin stimulation increased eNOS phosphorylation in non-diabetic subjects but not in diabetic patients (P=0.003) consistent with endothelial insulin resistance. Nitrotyrosine levels were higher in diabetic patients indicating endothelial oxidative stress. PKCβ expression was higher in diabetic patients and was associated with lower flow-mediated dilation (r=−0.541, P=0.02) Inhibition of PKCβ with LY379196 reduced basal eNOS phosphorylation and improved insulin-mediated eNOS activation in patients with diabetes. Endothelial NFκB activation was higher in diabetes and was reduced with PKCβ inhibition. Conclusions We provide evidence for the presence of altered eNOS activation, reduced insulin action and inflammatory activation in the endothelium of patients with diabetes. Our findings implicate PKCβ activity in endothelial insulin resistance. PMID:23204109

NFκB in the Mechanism of Ammonia-Induced Astrocyte Swelling in Culture

PubMed Central

Sinke, A.P.; Jayakumar, A.R.; Panickar, K.S.; Moriyama, M.; Reddy, P.V.B.; Norenberg, M.D.

2008-01-01

Astrocyte swelling and brain edema are major neuropathological findings in the acute form of hepatic encephalopathy (fulminant hepatic failure, FHF), and substantial evidence supports the view that elevated brain ammonia level is an important etiological factor in this condition. Although the mechanism by which ammonia brings about astrocyte swelling remains to be determined, oxidative/nitrosative stress and mitogen-activated protein kinases (MAPKs) have been considered as important elements in this process. One factor known to be activated by both oxidative stress and MAPKs is nuclear factor κB (NFκB), a transcription factor that activates many genes, including inducible nitric oxide synthase (iNOS). Since the product of iNOS, nitric oxide (NO), is known to cause astrocyte swelling, we examined the potential involvement of NFκB in ammonia-induced astrocyte swelling. Western blot analysis of cultured astrocytes showed a significant increase in NFκB nuclear translocation (a measure of NFκB activation) from 12 h to 2 days after treatment with NH4Cl (5 mM). Cultures treated with antioxidants, including superoxide dismutase, catalase and vitamin E, as well as the MAPKs inhibitors SB239063 (an inhibitor of p38-MAPK), and SP600125 (an inhibitor of c-Jun N-terminal kinase, JNK) significantly diminished NFκB activation by ammonia, supporting a role of oxidative stress and MAPKs in NFκB activation. The activation of NFκB was associated with increased iNOS protein expression and NO generation, and these changes were blocked by BAY 11-7082, an inhibitor of NFκB. Additionally, ammonia-induced astrocyte swelling was inhibited by the NFκB inhibitors BAY 11-7082 and SN-50, thereby implicating NFκB in the mechanism of astrocyte swelling. Our studies indicate that cultured astrocytes exposed to ammonia display NFκB activation, which is likely a consequence of oxidative stress and activation of MAPKs. NFκB activation appears to contribute to the mechanism of ammonia-induced astrocyte swelling, apparently through its upregulation of iNOS protein expression and the subsequent generation of nitric oxide. PMID:18662246

Essential role for uncoupling protein-3 in mitochondrial adaptation to fasting but not in fatty acid oxidation or fatty acid anion export.

PubMed

Seifert, Erin L; Bézaire, Véronic; Estey, Carmen; Harper, Mary-Ellen

2008-09-12

Uncoupling protein-3 (UCP3) is a mitochondrial inner membrane protein expressed most abundantly in skeletal muscle and to a lesser extent in heart and brown adipose tissue. Evidence supports a role for UCP3 in fatty acid oxidation (FAO); however, the underlying mechanism has not been explored. In 2001 we proposed a role for UCP3 in fatty acid export, leading to higher FAO rates (Himms-Hagen, J., and Harper, M. E. (2001) Exp. Biol. Med. (Maywood) 226, 78-84). Specifically, this widely held hypothesis states that during elevated FAO rates, UCP3 exports fatty acid anions, thereby maintaining mitochondrial co-enzyme A availability; reactivation of exported fatty acid anions would ultimately enable increased FAO. Here we tested mechanistic aspects of this hypothesis as well as its functional implications, namely increased FAO rates. Using complementary mechanistic approaches in mitochondria from wild-type and Ucp3(-/-) mice, we find that UCP3 is not required for FAO regardless of substrate type or supply rate covering a 20-fold range. Fatty acid anion export and reoxidation during elevated FAO, although present in skeletal muscle mitochondria, are independent of UCP3 abundance. Interestingly, UCP3 was found to be necessary for the fasting-induced enhancement of FAO rate and capacity, possibly via mitigated mitochondrial oxidative stress. Thus, although our observations indicate that UCP3 can impact FAO rates, the mechanistic basis is not via an integral function for UCP3 in the FAO machinery. Overall our data indicate a function for UCP3 in mitochondrial adaptation to perturbed cellular energy balance and integrate previous observations that have linked UCP3 to reduced oxidative stress and FAO.

PROTEOMIC IDENTIFICATION OF CARBONYLATED PROTEINS AND THEIR OXIDATION SITES

PubMed Central

Madian, Ashraf G.; Regnier, Fred E.

2011-01-01

Excessive oxidative stress leaves a protein carbonylation fingerprint in biological systems. Carbonylation is an irreversible post translational modification (PTM) that often leads to the loss of protein function and can be a component of multiple diseases. Protein carbonyl groups can be generated directly (by amino acids oxidation and the a-amidation pathway) or indirectly by forming adducts with lipid peroxidation products or glycation and advanced glycation end-products. Studies of oxidative stress are complicated by the low concentration of oxidation products and wide array of routes by which proteins are carbonylated. The development of new selection and enrichment techniques coupled with advances in mass spectrometry are allowing identification of hundreds of new carbonylated protein products from a broad range of proteins located at many sites in biological systems. The focus of this review is on the use of proteomics tools and methods to identify oxidized proteins along with specific sites of oxidative damage and the consequences of protein oxidation. PMID:20521848

Malnutrition-associated liver steatosis and ATP depletion is caused by peroxisomal and mitochondrial dysfunction.

PubMed

van Zutphen, Tim; Ciapaite, Jolita; Bloks, Vincent W; Ackereley, Cameron; Gerding, Albert; Jurdzinski, Angelika; de Moraes, Roberta Allgayer; Zhang, Ling; Wolters, Justina C; Bischoff, Rainer; Wanders, Ronald J; Houten, Sander M; Bronte-Tinkew, Dana; Shatseva, Tatiana; Lewis, Gary F; Groen, Albert K; Reijngoud, Dirk-Jan; Bakker, Barbara M; Jonker, Johan W; Kim, Peter K; Bandsma, Robert H J

2016-12-01

Severe malnutrition in young children is associated with signs of hepatic dysfunction such as steatosis and hypoalbuminemia, but its etiology is unknown. Peroxisomes and mitochondria play key roles in various hepatic metabolic functions including lipid metabolism and energy production. To investigate the involvement of these organelles in the mechanisms underlying malnutrition-induced hepatic dysfunction we developed a rat model of malnutrition. Weanling rats were placed on a low protein or control diet (5% or 20% of calories from protein, respectively) for four weeks. Peroxisomal and mitochondrial structural features were characterized using immunofluorescence and electron microscopy. Mitochondrial function was assessed using high-resolution respirometry. A novel targeted quantitative proteomics method was applied to analyze 47 mitochondrial proteins involved in oxidative phosphorylation, tricarboxylic acid cycle and fatty acid β-oxidation pathways. Low protein diet-fed rats developed hypoalbuminemia and hepatic steatosis, consistent with the human phenotype. Hepatic peroxisome content was decreased and metabolomic analysis indicated peroxisomal dysfunction. This was followed by changes in mitochondrial ultrastructure and increased mitochondrial content. Mitochondrial function was impaired due to multiple defects affecting respiratory chain complex I and IV, pyruvate uptake and several β-oxidation enzymes, leading to strongly reduced hepatic ATP levels. Fenofibrate supplementation restored hepatic peroxisome abundance and increased mitochondrial β-oxidation capacity, resulting in reduced steatosis and normalization of ATP and plasma albumin levels. Malnutrition leads to severe impairments in hepatic peroxisomal and mitochondrial function, and hepatic metabolic dysfunction. We discuss the potential future implications of our findings for the clinical management of malnourished children. Severe malnutrition in children is associated with metabolic disturbances that are poorly understood. In order to study this further, we developed a malnutrition animal model and found that severe malnutrition leads to an impaired function of liver mitochondria which are essential for energy production and a loss of peroxisomes, which are important for normal liver metabolic function. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Quantitative proteomic analysis reveals novel mitochondrial targets of estrogen deficiency in the aged female rat heart.

PubMed

Lancaster, T S; Jefferson, S J; Hunter, J Craig; Lopez, Veronica; Van Eyk, J E; Lakatta, E G; Korzick, D H

2012-10-17

The incidence of myocardial infarction rises sharply at menopause, implicating a potential role for estrogen (E(2)) loss in age-related increases in ischemic injury. We aimed to identify quantitative changes to the cardiac mitochondrial proteome of aging females, based on the hypothesis that E(2) deficiency exacerbates age-dependent disruptions in mitochondrial proteins. Mitochondria isolated from left ventricles of adult (6 mo) and aged (24 mo) F344 ovary-intact or ovariectomized (OVX) rats were labeled with 8plex isobaric tags for relative and absolute quantification (iTRAQ; n = 5-6/group). Groups studied were adult, adult OVX, aged, and aged OVX. In vivo coronary artery ligation and in vitro mitochondrial respiration studies were also performed in a subset of rats. We identified 965 proteins across groups and significant directional changes in 67 proteins of aged and/or aged OVX; 32 proteins were unique to aged OVX. Notably, only six proteins were similarly altered in adult OVX (voltage-dependent ion channel 1, adenine nucleotide translocator 1, cytochrome c oxidase subunits VIIc and VIc, catalase, and myosin binding protein C). Proteins affected by aging were primarily related to cellular metabolism, oxidative stress, and cell death. The largest change occurred in monoamine oxidase-A (MAO-A), a source of oxidative stress. While acute MAO-A inhibition induced mild uncoupling in aged mitochondria, reductions in infarct size were not observed. Age-dependent alterations in mitochondrial signaling indicate a highly selective myocardial response to E(2) deficiency. The combined proteomic and functional approaches described here offer possibility of new protein targets for experimentation and therapeutic intervention in the aged female population.

Assessing protein oxidation by inorganic nanoparticles with enzyme-linked immunosorbent assay (ELISA).

PubMed

Sun, Wenjie; Luna-Velasco, Antonia; Sierra-Alvarez, Reyes; Field, Jim A

2013-03-01

Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme-linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn(2)O(3), and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS-dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Copyright © 2012 Wiley Periodicals, Inc.

Impact of Interfacial Composition on Lipid and Protein Co-Oxidation in Oil-in-Water Emulsions Containing Mixed Emulisifers.

PubMed

Zhu, Zhenbao; Zhao, Cui; Yi, Jianhua; Liu, Ning; Cao, Yuangang; Decker, Eric A; McClements, David Julian

2018-05-02

The impact of interfacial composition on lipid and protein co-oxidation in oil-in-water emulsions containing a mixture of proteins and surfactants was investigated. The emulsions consisted of 5% v/v walnut oil, 0.5% w/v whey protein isolate (WPI), and 0 to 0.4% w/v Tween 20 (pH 3 and pH 7). The protein surface load, magnitude of the ξ-potential, and mean particle diameter of the emulsions decreased as the Tween 20 concentration was increased, indicating the whey proteins were displaced by this nonionic surfactant. The whey proteins were displaced from the lipid droplet surfaces more readily at pH 3 than at pH 7, which may have been due to differences in the conformation or interactions of the proteins at the droplet surfaces at different pH values. Emulsions stabilized by whey proteins alone had relatively low lipid oxidation rates when incubated in the dark at 45 °C for up to 8 days, as determined by measuring lipid hydroperoxides and 2-thiobarbituric acid-reactive substances (TBARS). Conversely, the whey proteins themselves were rapidly oxidized, as shown by carbonyl formation, intrinsic fluorescence, sulfhydryl group loss, and electrophoresis measurements. Displacement of whey proteins from the interface by Tween 20 reduced protein oxidation but promoted lipid oxidation. These results indicated that the adsorbed proteins were more prone to oxidation than the nonadsorbed proteins, and therefore, they could act as better antioxidants. Protein oxidation was faster, while lipid oxidation was slower at pH 3 than at pH 7, which was attributed to a higher antioxidant activity of whey proteins under acidic conditions. These results highlight the importance of interfacial composition and solution pH on the oxidative stability of emulsions containing mixed emulsifiers.

Mechanisms of oxidative stress, apoptosis, and autophagy involved in graphene oxide nanomaterial anti-osteosarcoma effect.

PubMed

Tang, Zhibing; Zhao, Lin; Yang, Zaixing; Liu, Zhaohui; Gu, Jia; Bai, Bing; Liu, Jinlian; Xu, Jiaying; Yang, Huilin

2018-01-01

Graphene and its derivative graphene oxide (GO) have been implicated in a wide range of anticancer effects. The objective of this study was to systematically evaluate the toxicity and underlying mechanisms of GO on two osteosarcoma (OSA) cancer cell lines, MG-63 and K 7 M 2 cells. MG-63 and K 7 M 2 cells were treated by GO (0-50 µg/mL) for various time periods. Cell viability was tested by MTT and Live/Dead assays. A ROS Detection Kit based on DHE oxidative reaction was used for ROS detection. An Annexin V-FITC Apoptosis Kit was used for apoptosis detection. Dansylcadaverine (MDC) dyeing was applied for seeking unspecific autophagosomes. Western blot and Immunofluorescence analysis were used for related protein expression and location. K 7 M 2 cells were more sensitive to GO compared with MG-63 cells. The mechanism was attributed to the different extent of the generation of reactive oxygen species (ROS). In K 7 M 2 cells, ROS was easily stimulated and the apoptosis pathway was subsequently activated, accompanied by elevated expression of proapoptosis proteins (such as caspase-3) and decreased expression levels of antiapoptosis proteins (such as Bcl-2). A ROS inhibitor ( N -acetylcysteine) could alleviate the cytotoxic effects of GO in K 7 M 2 cells. However, the production of ROS in MG-63 cells was probably inhibited by the activation of an antioxidative factor, nuclear factor-E2-related factor-2, which translocated from the cytoplasm to the nucleus after GO treatment, while a nuclear factor-E2-related factor-2 inhibitor (ML385) significantly increased ROS production in MG-63 cells when combined with GO treatment. In addition, autophagy was simultaneously stimulated by characteristic autophagosome formation, autophagy flux, and increased the expression level of autophagy-related proteins (such as LC3I to LC3II conversion, ATG5, and ATG7). This paper proposes various underlying mechanisms of the anticancer effect of GO. The novel synthetic use of GO with an oxidizing agent is the key step for further potential applications in clinical OSA cancer therapy.

Analysis of Cysteine Redox Post-Translational Modifications in Cell Biology and Drug Pharmacology.

PubMed

Wani, Revati; Murray, Brion W

2017-01-01

Reversible cysteine oxidation is an emerging class of protein post-translational modification (PTM) that regulates catalytic activity, modulates conformation, impacts protein-protein interactions, and affects subcellular trafficking of numerous proteins. Redox PTMs encompass a broad array of cysteine oxidation reactions with different half-lives, topographies, and reactivities such as S-glutathionylation and sulfoxidation. Recent studies from our group underscore the lesser known effect of redox protein modifications on drug binding. To date, biological studies to understand mechanistic and functional aspects of redox regulation are technically challenging. A prominent issue is the lack of tools for labeling proteins oxidized to select chemotype/oxidant species in cells. Predictive computational tools and curated databases of oxidized proteins are facilitating structural and functional insights into regulation of the network of oxidized proteins or redox proteome. In this chapter, we discuss analytical platforms for studying protein oxidation, suggest computational tools currently available in the field to determine redox sensitive proteins, and begin to illuminate roles of cysteine redox PTMs in drug pharmacology.

Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System.

PubMed

Ozer, Hatice K; Dlouhy, Adrienne C; Thornton, Jeremy D; Hu, Jingjing; Liu, Yilin; Barycki, Joseph J; Balk, Janneke; Outten, Caryn E

2015-11-13

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidative Stress and Mitochondrial Functions in the Intestinal Caco-2/15 Cell Line

PubMed Central

Taha, Rame; Seidman, Ernest; Mailhot, Genevieve; Boudreau, François; Gendron, Fernand-Pierre; Beaulieu, Jean-François; Ménard, Daniel; Delvin, Edgard; Amre, Devendra; Levy, Emile

2010-01-01

Background Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules. Methodology/Principal Findings The objective of the present work was to evaluate how iron-ascorbate (FE/ASC)-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM) (1) increased malondialdehyde levels assessed by HPLC; (2) reduced ATP production noted by luminescence assay; (3) provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4) upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5) affected mitochondrial respiratory chain complexes I, II, III and IV; (6) elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7) lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8) altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2) without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 µM) prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities. Conclusions/Significance Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases. PMID:20676402

Oxidative stress and mitochondrial functions in the intestinal Caco-2/15 cell line.

PubMed

Taha, Rame; Seidman, Ernest; Mailhot, Genevieve; Boudreau, François; Gendron, Fernand-Pierre; Beaulieu, Jean-François; Ménard, Daniel; Delvin, Edgard; Amre, Devendra; Levy, Emile

2010-07-27

Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules. The objective of the present work was to evaluate how iron-ascorbate (FE/ASC)-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM) (1) increased malondialdehyde levels assessed by HPLC; (2) reduced ATP production noted by luminescence assay; (3) provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4) upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5) affected mitochondrial respiratory chain complexes I, II, III and IV; (6) elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7) lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8) altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2) without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 microM) prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities. Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases.

The role of the Golgi apparatus in oxidative stress: is this organelle less significant than mitochondria?

PubMed

Jiang, Zheng; Hu, Zhiping; Zeng, Liuwang; Lu, Wei; Zhang, Hainan; Li, Ting; Xiao, Han

2011-04-15

Reactive oxygen species (ROS)/reactive nitrogen species (RNS) and ROS/RNS-mediated oxidative stress have well-established roles in many physiological and pathological processes and are associated with the pathogenesis of many diseases, such as hypertension, ischemia/reperfusion injury, diabetes mellitus, atherosclerosis, stroke, cancer, and neurodegenerative disorders. It is generally accepted that mitochondria play an essential role in oxidative stress because they are responsible for the primary generation of superoxide radicals. Little attention, however, has been paid to the importance of the Golgi apparatus (GA) in this process. The GA is a pivotal organelle in cell metabolism and participates in modifying, sorting, and packaging macromolecules for cell secretion or use within the cell. It is inevitably involved in the process of oxidative stress, which can cause modification and damage of lipids, proteins, DNA, and other structural constituents. Here we discuss the connections between the GA and oxidative stress and highlight the role of the GA in oxidative stress-related Ca(2+)/Mn(2+) homeostasis, cell apoptosis, sphingolipid metabolism, signal transduction, and antioxidation. We also provide a novel perspective on the subcellular significance of oxidative stress and its pathological implications and present "GA stress" as a new concept to explain the GA-specific stress response. Copyright © 2011 Elsevier Inc. All rights reserved.

The Measurement of Reversible Redox Dependent Post-translational Modifications and Their Regulation of Mitochondrial and Skeletal Muscle Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kramer, Philip A.; Duan, Jicheng; Qian, Wei-Jun

Mitochondrial oxidative stress is a common feature of skeletal myopathies across multiple conditions; however, the mechanism by which it contributes to skeletal muscle dysfunction remains controversial. Oxidative damage to proteins, lipids, and DNA has received the most attention, yet an important role for reversible redox post-translational modifications (PTMs) in pathophysiology is emerging. The possibility that these PTMs can exert dynamic control of muscle function implicates them as a mechanism contributing to skeletal muscle dysfunction in chronic disease. Herein, we discuss the significance of thiol-based redox dependent modifications to mitochondrial, myofibrillar and excitation-contraction (EC) coupling proteins with an emphasis on howmore » these changes could alter skeletal muscle performance under chronically stressed conditions. A major barrier to a better mechanistic understanding of the role of reversible redox PTMs in muscle function is the technical challenges associated with accurately measuring the changes of site-specific redox PTMs. Here we will critically review current approaches with an emphasis on sample preparation artifacts, quantitation, and specificity. Despite these challenges, the ability to accurately quantify reversible redox PTMs is critical to understanding the mechanisms by which mitochondrial oxidative stress contributes to skeletal muscle dysfunction in chronic diseases.« less

Oxidative stress generated during monensin treatment contributes to altered Toxoplasma gondii mitochondrial function

PubMed Central

Charvat, Robert A.; Arrizabalaga, Gustavo

2016-01-01

The ionophore monensin displays potent activities against several coccidian parasites of veterinary and medical importance including the opportunistic pathogen of humans, Toxoplasma gondii. While monensin is used widely in animals, toxicity impedes its use in humans. Nonetheless, given its potency, understanding its mode of action would reveal vulnerable aspects of the parasite that can be exploited for drug development. We previously established that monensin induces Toxoplasma to undergo cell cycle arrest and an autophagy-like cell death. Interestingly, these effects are dependent on the mitochondrion-localized TgMSH-1 protein, suggesting that monensin disrupts mitochondrial function. We demonstrate that monensin treatment results in decreased mitochondrial membrane potential and altered morphology. These effects are mitigated by the antioxidant compound N-acetyl-cysteine suggesting that monensin causes an oxidative stress, which was indeed the case based on direct detection of reactive oxygen species. Moreover, over-expression of the antioxidant proteins glutaredoxin and peroxiredoxin 2 protect Toxoplasma from the deleterious effects of monensin. Thus, our studies show that the effects of monensin on Toxoplasma are due to a disruption of mitochondrial function caused by the induction of an oxidative stress and implicate parasite redox biology as a viable target for the development of drugs against Toxoplasma and related pathogenic parasites. PMID:26976749

Loss of NEIL3 DNA glycosylase markedly increases replication associated double strand breaks and enhances sensitivity to ATR inhibitor in glioblastoma cells

PubMed Central

Klattenhoff, Alex W.; Thakur, Megha; Chu, Christopher S.; Ray, Debolina; Habib, Samy L.; Kidane, Dawit

2017-01-01

DNA endonuclease eight-like glycosylase 3 (NEIL3) is one of the DNA glycosylases that removes oxidized DNA base lesions from single-stranded DNA (ssDNA) and non-B DNA structures. Approximately seven percent of human tumors have an altered NEIL3 gene. However, the role of NEIL3 in replication-associated repair and its impact on modulating treatment response is not known. Here, we report that NEIL3 is localized at the DNA double-strand break (DSB) sites during oxidative DNA damage and replication stress. Loss of NEIL3 significantly increased spontaneous replication-associated DSBs and recruitment of replication protein A (RPA). In contrast, we observed a marked decrease in Rad51 on nascent DNA strands at the replication fork, suggesting that HR-dependent repair is compromised in NEIL3-deficient cells. Interestingly, NEIL3-deficient cells were sensitive to ataxia–telangiectasia and Rad3 related protein (ATR) inhibitor alone or in combination with PARP1 inhibitor. This study elucidates the mechanism by which NEIL3 is critical to overcome oxidative and replication-associated genotoxic stress. Our findings may have important clinical implications to utilize ATR and PARP1 inhibitors to enhance cytotoxicity in tumors that carry altered levels of NEIL3. PMID:29348879

Skeletal muscle action of estrogen receptor α is critical for the maintenance of mitochondrial function and metabolic homeostasis in females.

PubMed

Ribas, Vicent; Drew, Brian G; Zhou, Zhenqi; Phun, Jennifer; Kalajian, Nareg Y; Soleymani, Teo; Daraei, Pedram; Widjaja, Kevin; Wanagat, Jonathan; de Aguiar Vallim, Thomas Q; Fluitt, Amy H; Bensinger, Steven; Le, Thuc; Radu, Caius; Whitelegge, Julian P; Beaven, Simon W; Tontonoz, Peter; Lusis, Aldons J; Parks, Brian W; Vergnes, Laurent; Reue, Karen; Singh, Harpreet; Bopassa, Jean C; Toro, Ligia; Stefani, Enrico; Watt, Matthew J; Schenk, Simon; Akerstrom, Thorbjorn; Kelly, Meghan; Pedersen, Bente K; Hewitt, Sylvia C; Korach, Kenneth S; Hevener, Andrea L

2016-04-13

Impaired estrogen receptor α (ERα) action promotes obesity and metabolic dysfunction in humans and mice; however, the mechanisms underlying these phenotypes remain unknown. Considering that skeletal muscle is a primary tissue responsible for glucose disposal and oxidative metabolism, we established that reduced ERα expression in muscle is associated with glucose intolerance and adiposity in women and female mice. To test this relationship, we generated muscle-specific ERα knockout (MERKO) mice. Impaired glucose homeostasis and increased adiposity were paralleled by diminished muscle oxidative metabolism and bioactive lipid accumulation in MERKO mice. Aberrant mitochondrial morphology, overproduction of reactive oxygen species, and impairment in basal and stress-induced mitochondrial fission dynamics, driven by imbalanced protein kinase A-regulator of calcineurin 1-calcineurin signaling through dynamin-related protein 1, tracked with reduced oxidative metabolism in MERKO muscle. Although muscle mitochondrial DNA (mtDNA) abundance was similar between the genotypes, ERα deficiency diminished mtDNA turnover by a balanced reduction in mtDNA replication and degradation. Our findings indicate the retention of dysfunctional mitochondria in MERKO muscle and implicate ERα in the preservation of mitochondrial health and insulin sensitivity as a defense against metabolic disease in women. Copyright © 2016, American Association for the Advancement of Science.

MutY: optimized to find DNA damage site electronically?

NASA Astrophysics Data System (ADS)

Lin, Jong-Chin; Cox, Daniel; Singh, Rajiv

2006-03-01

Iron sulfur clusters are present in the DNA repair protein MutY in a region highly homologous in species as diverse as E. Coli and Homo Sapiens, yet their function remains unknown. In MutY, this mixed valence cluster exists in two oxidation states, [Fe4S4]^2+/3+, with the stability depending upon the presence of DNA. We have studied the electronic structure and stability of these clusters using the local orbital based SIESTA implementation of density functional theory. We find that the iron-sulfur cluster in MutY can undergo 2+ to 3+ oxidation when coupling to DNA through hole transfer, especially when MutY is near an oxoguanine modified base(oxoG). Employing the Marcus theory for electron transfer, we find (i) near optimal Frank-Condon(FC) factor for 2+ transfer to oxoG; (ii) reduced FC factor for transfer to G due to a high oxidation potential; (iii) reduced FC factor with the mutation L154F; (iv) reduced tunning matrix element with the mutation R149W. Both L154F and R149W mutations dramatically reduce or eliminate repair efficiency. Hence, redox modulation of MutY search and binding appears plausible and may have broader implications for DNA-protein interactions.

Reactive metabolites and antioxidant gene polymorphisms in type 2 diabetes mellitus

PubMed Central

Banerjee, Monisha; Vats, Pushpank

2014-01-01

Type 2 diabetes mellitus (T2DM), by definition is a heterogeneous, multifactorial, polygenic syndrome which results from insulin receptor (IR) dysfunction. It is an outcome of oxidative stress caused by interactions of reactive metabolites (RMs) with lipids, proteins and other molecules of the human body. Production of RMs mainly superoxides (•O2−) has been found in a variety of predominating cellular enzyme systems including nicotinamide adenine dinucleotide phosphate oxidase, xanthine oxidase, cyclooxygenase, endothelial nitric oxide synthase (eNOS) and myeloperoxidase. The four main RM related molecular mechanisms are: increased polyol pathway flux; increased advanced glycation end-product formation; activation of protein kinase C isoforms and increased hexosamine pathway flux which have been implicated in glucose-mediated vascular damage. Superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and NOS are antioxidant enzymes involved in scavenging RMs in normal individuals. Functional polymorphisms of these antioxidant enzymes have been reported to be involved in the pathogenesis of T2DM. The low levels of antioxidant enzymes or their non-functionality results in excessive RMs which initiates stress related pathways thereby leading to IR and T2DM. An attempt has been made to review the role of RMs and antioxidant enzymes in oxidative stress resulting in T2DM. PMID:24959009

Nickel inhibits mitochondrial fatty acid oxidation.

PubMed

Uppala, Radha; McKinney, Richard W; Brant, Kelly A; Fabisiak, James P; Goetzman, Eric S

2015-08-07

Nickel exposure is associated with changes in cellular energy metabolism which may contribute to its carcinogenic properties. Here, we demonstrate that nickel strongly represses mitochondrial fatty acid oxidation-the pathway by which fatty acids are catabolized for energy-in both primary human lung fibroblasts and mouse embryonic fibroblasts. At the concentrations used, nickel suppresses fatty acid oxidation without globally suppressing mitochondrial function as evidenced by increased glucose oxidation to CO2. Pre-treatment with l-carnitine, previously shown to prevent nickel-induced mitochondrial dysfunction in neuroblastoma cells, did not prevent the inhibition of fatty acid oxidation. The effect of nickel on fatty acid oxidation occurred only with prolonged exposure (>5 h), suggesting that direct inhibition of the active sites of metabolic enzymes is not the mechanism of action. Nickel is a known hypoxia-mimetic that activates hypoxia inducible factor-1α (HIF1α). Nickel-induced inhibition of fatty acid oxidation was blunted in HIF1α knockout fibroblasts, implicating HIF1α as one contributor to the mechanism. Additionally, nickel down-regulated the protein levels of the key fatty acid oxidation enzyme very long-chain acyl-CoA dehydrogenase (VLCAD) in a dose-dependent fashion. In conclusion, inhibition of fatty acid oxidation by nickel, concurrent with increased glucose metabolism, represents a form of metabolic reprogramming that may contribute to nickel-induced carcinogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Influence of chirality on catalytic generation of nitric oxide and platelet behavior on selenocystine immobilized TiO2 films.

PubMed

Fan, Yonghong; Pan, Xiaxin; Wang, Ke; Wu, Sisi; Han, Honghong; Yang, Ping; Luo, Rifang; Wang, Hong; Huang, Nan; Tan, Wei; Weng, Yajun

2016-09-01

As nitric oxide (NO) plays vital roles in the cardiovascular system, incorporating this molecule into cardiovascular stents is considered as an effective method. In the present study, selenocystine with different chirality (i.e., l- and d-selenocystine) was used as the catalytic molecule immobilized on TiO2 films for decomposing endogenous NO donor. The influences of surface chirality on NO release and platelet behavior were evaluated. Results show that although the amount of immobilized l-selenocystine on the surface was nearly the same as that of immobilized d-selenocystine, in vitro catalytic NO release tests showed that l-selenocystine immobilized surfaces were more capable of catalyzing the decomposition of S-nitrosoglutathione and thus generating more NO. Accordingly, l-selenocystine immobilized surfaces demonstrated significantly increased inhibiting effects on the platelet adhesion and activation, when compared to d-selenocystine immobilized ones. Measurement of the cGMP concentration of platelets further confirmed that surface chirality played an important role in regulating NO generation and platelet behaviors. Additionally, using bovine serum albumin and fibrinogen as model proteins, the protein adsorption determined with quartz crystal microbalance showed that the l-selenocystine immobilized surface enhanced protein adsorption. In conclusion, surface chirality significantly influences protein adsorption and NO release, which may have significant implications in the design of NO-generating cardiovascular stents. Copyright © 2016 Elsevier B.V. All rights reserved.

Environmental Pollutant Ozone Causes Damage to Lung Surfactant Protein B (SP-B)

PubMed Central

2015-01-01

Lung surfactant protein B (SP-B) is an essential protein found in the surfactant fluid at the air–water interface of the lung. Exposure to the air pollutant ozone could potentially damage SP-B and lead to respiratory distress. We have studied two peptides, one consisting of the N-terminus of SP-B [SP-B(1–25)] and the other a construct of the N- and C-termini of SP-B [SP-B(1–25,63–78)], called SMB. Exposure to dilute levels of ozone (∼2 ppm) of monolayers of each peptide at the air–water interface leads to a rapid reaction, which is evident from an increase in the surface tension. Fluorescence experiments revealed that this increase in surface tension is accompanied by a loss of fluorescence from the tryptophan residue at the interface. Neutron and X-ray reflectivity experiments show that, in contrast to suggestions in the literature, the peptides are not solubilized upon oxidation but rather remain at the interface with little change in their hydration. Analysis of the product material reveals that no cleavage of the peptides occurs, but a more hydrophobic product is slowly formed together with an increased level of oligomerization. We attributed this to partial unfolding of the peptides. Experiments conducted in the presence of phospholipids reveal that the presence of the lipids does not prevent oxidation of the peptides. Our results strongly suggest that exposure to low levels of ozone gas will damage SP-B, leading to a change in its structure. The implication is that the oxidized protein will be impaired in its ability to interact at the air–water interface with negatively charged phosphoglycerol lipids, thus compromising what is thought to be its main biological function. PMID:26270023

Transitions towards either slow-oxidative or fast-glycolytic phenotype can be induced in the murine WTt myogenic cell line.

PubMed

Peltzer, J; Carpentier, G; Martelly, I; Courty, J; Keller, A

2010-09-01

Contraction and energy metabolism are functions of skeletal muscles co-regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow-oxidative or fast-glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF-1alpha and PPARdelta. Our data support the idea that HIF-1alpha protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARdelta over-expression appears to enhance the slow-oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC-1alpha, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF-1alpha protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF-1alpha and PPARdelta. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF-1alpha and PPARdelta in the coordinated metabolic and contractile regulations involved in myogenesis. (c) 2010 Wiley-Liss, Inc.

Alterations in Glutathione Redox Metabolism, Oxidative Stress, and Mitochondrial Function in the Left Ventricle of Elderly Zucker Diabetic Fatty Rat Heart

PubMed Central

Raza, Haider; John, Annie; Howarth, Frank C.

2012-01-01

The Zucker diabetic fatty (ZDF) rat is a genetic model in which the homozygous (FA/FA) male animals develop obesity and type 2 diabetes. Morbidity and mortality from cardiovascular complications, due to increased oxidative stress and inflammatory signals, are the hallmarks of type 2 diabetes. The precise molecular mechanism of contractile dysfunction and disease progression remains to be clarified. Therefore, we have investigated molecular and metabolic targets in male ZDF (30–34 weeks old) rat heart compared to age matched Zucker lean (ZL) controls. Hyperglycemia was confirmed by a 4-fold elevation in non-fasting blood glucose (478.43 ± 29.22 mg/dL in ZDF vs. 108.22 ± 2.52 mg/dL in ZL rats). An increase in reactive oxygen species production, lipid peroxidation and oxidative protein carbonylation was observed in ZDF rats. A significant increase in CYP4502E1 activity accompanied by increased protein expression was also observed in diabetic rat heart. Increased expression of other oxidative stress marker proteins, HO-1 and iNOS was also observed. GSH concentration and activities of GSH-dependent enzymes, glutathione S-transferase and GSH reductase, were, however, significantly increased in ZDF heart tissue suggesting a compensatory defense mechanism. The activities of mitochondrial respiratory enzymes, Complex I and Complex IV were significantly reduced in the heart ventricle of ZDF rats in comparison to ZL rats. Western blot analysis has also suggested a decreased expression of IκB-α and phosphorylated-JNK in diabetic heart tissue. Our results have suggested that mitochondrial dysfunction and increased oxidative stress in ZDF rats might be associated, at least in part, with altered NF-κB/JNK dependent redox cell signaling. These results might have implications in the elucidation of the mechanism of disease progression and designing strategies for diabetes prevention. PMID:23203193

Influence of sodium nitrite on protein oxidation and nitrosation of sausages subjected to processing and storage.

PubMed

Feng, Xianchao; Li, Chenyi; Jia, Xu; Guo, Yan; Lei, Na; Hackman, Robert M; Chen, Lin; Zhou, Guanghong

2016-06-01

The influence of NaNO2 content on protein oxidation and nitrosation was investigated in cooked sausages at different concentrations (0, 50, 100, 200 and 400 mg NaNO2/kg). Dependent on concentration, NaNO2 had both anti- and pro-oxidant effects on protein oxidation. The antioxidant effects of NaNO2 on the protein oxidation were evidenced by significantly lower carbonyl contents, higher free amines and lower surface hydrophobicities. The pro-oxidant effects of NaNO2 on protein oxidation resulted in a decrease of sulfhydryls and an increase of disulfide bonds. NaNO2 also improved the protein nitrosation inducing the formation of 3-nitrotyrosine (3-NT). Moreover, 3-NT had significant correlations with parameters of protein oxidation, indicating that 3-NT may be a possible marker for protein oxidation. Results of this study contribute to an understanding of the impact of NaNO2 on food quality and help to identify optimal formulations of cured meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Studies on glycoxidatively modified human IgG: Implications in immuno-pathology of type 2 diabetes mellitus.

PubMed

Islam, Sidra; Moinuddin; Mir, Abdul Rouf; Arfat, Mir Yasir; Alam, Khursheed; Ali, Asif

2017-11-01

Structural rearrangements and condensations of proteins under hyperglycemic stress have been implicated in various pathological disorders. This study aims to probe the role of methylglyoxal (MG) modified human immunoglobulin G (MG-IgG) in immuno-pathology of type 2 diabetes mellitus (T2DM). MG was found to perturb the structural integrity of IgG, affect its aromatic micro-environment and cause the generation of advanced glycation end products (AGEs) and aggregate adducts. It liberated the hydrophobic pockets of the protein, reduced its β pleated sheet structure and affected its tertiary conformation. Transition from β sheet to α helix and random coil was also observed in IgG upon modification by MG. It acted with strong oxidative potential and caused oligomerisation and disordered or amorphous type aggregation in the modified protein. Modified IgG had a cytotoxic and genotoxic impact. The MG modified IgG presented novel antigenic determinants that lead to an aggressive immune response. The antibodies had high affinity towards the immunogen. Auto-antibodies derived from T2DM patients exhibited strong affinity towards the modified IgG in comparison to the unmodified protein. Specificity of serum antibodies from T2DM patients was further confirmed by competitive-inhibition ELISA. The potential role of MG-IgG in the immunopathogenesis of T2DM has been discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

The Role of Stress-Induced O-GlcNAc Protein Modification in the Regulation of Membrane Transport

PubMed Central

Fisi, Viktória; Miseta, Attila

2017-01-01

O-linked N-acetylglucosamine (O-GlcNAc) is a posttranslational modification that is increasingly recognized as a signal transduction mechanism. Unlike other glycans, O-GlcNAc is a highly dynamic and reversible process that involves the addition and removal of a single N-acetylglucosamine molecule to Ser/Thr residues of proteins. UDP-GlcNAc—the direct substrate for O-GlcNAc modification—is controlled by the rate of cellular metabolism, and thus O-GlcNAc is dependent on substrate availability. Serving as a feedback mechanism, O-GlcNAc influences the regulation of insulin signaling and glucose transport. Besides nutrient sensing, O-GlcNAc was also implicated in the regulation of various physiological and pathophysiological processes. Due to improvements of mass spectrometry techniques, more than one thousand proteins were detected to carry the O-GlcNAc moiety; many of them are known to participate in the regulation of metabolites, ions, or protein transport across biological membranes. Recent studies also indicated that O-GlcNAc is involved in stress adaptation; overwhelming evidences suggest that O-GlcNAc levels increase upon stress. O-GlcNAc elevation is generally considered to be beneficial during stress, although the exact nature of its protective effect is not understood. In this review, we summarize the current data regarding the oxidative stress-related changes of O-GlcNAc levels and discuss the implications related to membrane trafficking. PMID:29456783

Protein Oxidation in Aging: Does It Play a Role in Aging Progression?

PubMed Central

Reeg, Sandra

2015-01-01

Abstract Significance: A constant accumulation of oxidized proteins takes place during aging. Oxidation of proteins leads to a partial unfolding and, therefore, to aggregation. Protein aggregates impair the activity of cellular proteolytic systems (proteasomes, lysosomes), resulting in further accumulation of oxidized proteins. In addition, the accumulation of highly crosslinked protein aggregates leads to further oxidant formation, damage to macromolecules, and, finally, to apoptotic cell death. Furthermore, protein oxidation seems to play a role in the development of various age-related diseases, for example, neurodegenerative diseases. Recent Advances: The highly oxidized lipofuscin accumulates during aging. Lipofuscin formation might cause impaired lysosomal and proteasomal degradation, metal ion accumulation, increased reactive oxygen species formation, and apoptosis. Critical Issues: It is still unclear to which extent protein oxidation is involved in the progression of aging and in the development of some age-related diseases. Future Directions: An extensive knowledge of the effects of protein oxidation on the aging process and its contribution to the development of age-related diseases could enable further strategies to reduce age-related impairments. Strategies aimed at lowering aggregate formation might be a straightforward intervention to reduce age-related malfunctions of organs. Antioxid. Redox Signal. 23, 239–255. PMID:25178482

A double stain for total and oxidized proteins from two-dimensional fingerprints.

PubMed

Talent, J M; Kong, Y; Gracy, R W

1998-10-01

Oxidative modification of proteins plays a major role in the etiology of aging and age-related diseases. For example, in Alzheimer's disease, although evidence points to oxidation of proteins as a causative factor in loss of cognitive abilities, it is not known which specific proteins of the brain are most susceptible to these modifications. Thus, it is of interest to identify the specific proteins which are susceptible to oxidation in vivo. Two-dimensional protein fingerprint methods offer the analytical potential for resolution of thousands of individual proteins from tissues, and the oxidized proteins can be visualized with immunological probes. Sensitive methods permit recovery and sufficient amino acid sequencing to identify these proteins. However, for such analyses it is essential to simultaneously analyze both protein content and level of oxidation. We have evaluated several approaches, identified the sources of artifacts and interferences, and developed a double-staining procedure that allows visualization and quantitation of total protein patterns as well as the specific oxidized proteins from two-dimensional protein fingerprints. The method has been applied to cells grown in culture and to tissue extracts from young and old animals. Copyright 1998 Academic Press.

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging*

PubMed Central

Ramallo Guevara, Carina; Philipp, Oliver; Hamann, Andrea; Werner, Alexandra; Osiewacz, Heinz D.; Rexroth, Sascha; Rögner, Matthias; Poetsch, Ansgar

2016-01-01

The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age. PMID:26884511

Age-related changes in HSP25 expression in basal ganglia and cortex of F344/BN rats

PubMed Central

Gupte, Anisha A.; Morris, Jill K.; Zhang, Hongyu; Bomhoff, Gregory L.; Geiger, Paige C.; Stanford, John A.

2010-01-01

Normal aging is associated with chronic oxidative stress. In the basal ganglia, oxidative stress may contribute to the increased risk of Parkinson's disease in the elderly. Neurons are thought to actively utilize compensatory defense mechanisms, such as heat shock proteins (HSPs), to protect from persisting stress. Despite their protective role, little is known about HSP expression in the aging basal ganglia. The purpose of this study was to examine HSP expression in striatum, substantia nigra, globus pallidus and cortex in 6-, 18- and 30-month-old Fischer 344/Brown Norway rats. We found robust age-related increases in phosphorylated and total HSP25 in each brain region studied. Conversely, HSP72 (the inducible form of HSP70) was reduced with age, but only in the striatum. p38 MAPK, a protein implicated in activating HSP25, did not change with age, nor did HSC70 (the constitutive form of HSP70), or HSP60. These results suggest that HSP25 is especially responsive to age-related stress in the basal ganglia. PMID:20144690

Novel Directions for Diabetes Mellitus Drug Discovery

PubMed Central

Maiese, Kenneth; Chong, Zhao Zhong; Shang, Yan Chen; Wang, Shaohui

2012-01-01

Introduction Diabetes mellitus impacts almost 200 million individuals worldwide and leads to debilitating complications. New avenues of drug discovery must target the underlying cellular processes of oxidative stress, apoptosis, autophagy, and inflammation that can mediate multi-system pathology during diabetes mellitus. Areas Covered We examine novel directions for drug discovery that involve the β-nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide, the cytokine erythropoietin, the NAD+-dependent protein histone deacetylase SIRT1, the serine/threonine-protein kinase mammalian target of rapamycin (mTOR), and the wingless pathway. Implications for the targeting of these pathways that oversee gluconeogenic genes, insulin signaling and resistance, fatty acid beta-oxidation, inflammation, and cellular survival are presented. Expert Opinion Nicotinamide, erythropoietin, and the downstram pathways of SIRT1, mTOR, forkhead transcription factors, and wingless signaling offer exciting prospects for novel directions of drug discovery for the treatment of metabolic disorders. Future investigations must dissect the complex relationship and fine modulation of these pathways for the successful translation of robust reparative and regenerative strategies against diabetes mellitus and the complications of this disorder. PMID:23092114

Metagenomic Evidence for the Presence of Comammox Nitrospira-Like Bacteria in a Drinking Water System.

PubMed

Pinto, Ameet J; Marcus, Daniel N; Ijaz, Umer Zeeshan; Bautista-de Lose Santos, Quyen Melina; Dick, Gregory J; Raskin, Lutgarde

2016-01-01

We report metagenomic evidence for the presence of a Nitrospira-like organism with the metabolic potential to perform the complete oxidation of ammonia to nitrate (i.e., it is a complete ammonia oxidizer [comammox]) in a drinking water system. This metagenome bin was discovered through shotgun DNA sequencing of samples from biologically active filters at the drinking water treatment plant in Ann Arbor, MI. Ribosomal proteins, 16S rRNA, and nxrA gene analyses confirmed that this genome is related to Nitrospira-like nitrite-oxidizing bacteria. The presence of the full suite of ammonia oxidation genes, including ammonia monooxygenase and hydroxylamine dehydrogenase, on a single ungapped scaffold within this metagenome bin suggests the presence of recently discovered comammox potential. Evaluations based on coverage and k-mer frequency distribution, use of two different genome-binning approaches, and nucleic acid and protein similarity analyses support the presence of this scaffold within the Nitrospira metagenome bin. The amoA gene found in this metagenome bin is divergent from those of canonical ammonia and methane oxidizers and clusters closely with the unusual amoA gene of comammox Nitrospira. This finding suggests that previously reported imbalances in abundances of nitrite- and ammonia-oxidizing bacteria/archaea may likely be explained by the capacity of Nitrospira-like organisms to completely oxidize ammonia. This finding might have significant implications for our understanding of microbially mediated nitrogen transformations in engineered and natural systems. IMPORTANCE Nitrification plays an important role in regulating the concentrations of inorganic nitrogen species in a range of environments, from drinking water and wastewater treatment plants to the oceans. Until recently, aerobic nitrification was considered to be a two-step process involving ammonia-oxidizing bacteria or archaea and nitrite-oxidizing bacteria. This process requires close cooperation between these two functional guilds for complete conversion of ammonia to nitrate, without the accumulation of nitrite or other intermediates, such as nitrous oxide, a potent greenhouse gas. The discovery of a single organism with the potential to oxidize both ammonia and nitrite adds a new dimension to the current understanding of aerobic nitrification, while presenting opportunities to rethink nitrogen management in engineered systems.

Oxidative tyrosylation of high density lipoprotein by peroxidase enhances cholesterol removal from cultured fibroblasts and macrophage foam cells.

PubMed Central

Francis, G A; Mendez, A J; Bierman, E L; Heinecke, J W

1993-01-01

Lipoprotein oxidation is thought to play a pivotal role in atherogenesis, yet the underlying reaction mechanisms remain poorly understood. We have explored the possibility that high density lipoprotein (HDL) might be oxidized by peroxidase-generated tyrosyl radical. Exposure of HDL to L-tyrosine, H2O2, and horseradish peroxidase crosslinked its apolipoproteins and strikingly increased protein-associated fluorescence. The reaction required L-tyrosine but was independent of free metal ions; it was blocked by either catalase or the heme poison aminotriazole. Dityrosine and other tyrosine oxidation products were detected in the apolipoproteins of HDL modified by the peroxidase/L-tyrosine/H2O2 system, implicating tyrosyl radical in the reaction pathway. Further evidence suggests that tyrosylated HDL removes cholesterol from cultured cells more effectively than does HDL. Tyrosylated HDL was more potent than HDL at inhibiting cholesterol esterification by the acyl-CoA:cholesterol acyltransferase reaction, stimulating the incorporation of [14C]acetate into [14C]cholesterol, and depleting cholesteryl ester stores in human skin fibroblasts. Moreover, exposure of mouse macrophage foam cells to tyrosylated HDL markedly diminished cholesteryl ester and free cholesterol mass. We have recently found that myeloperoxidase, a heme protein secreted by activated phagocytes, can also convert L-tyrosine to o,o'-dityrosine. This raises the possibility that myeloperoxidase-generated tyrosyl radical may modify HDL, enabling the lipoprotein to protect the artery wall against pathological cholesterol accumulation. Images Fig. 1 PMID:8341680

Excess TGF-β mediates muscle weakness associated with bone metastases in mice

PubMed Central

Reiken, Steven; Xie, Wenjun; Andersson, Daniel C.; John, Sutha; Chiechi, Antonella; Wright, Laura E.; Umanskaya, Alisa; Niewolna, Maria; Trivedi, Trupti; Charkhzarrin, Sahba; Khatiwada, Pooja; Wronska, Anetta; Haynes, Ashley; Benassi, Maria Serena; Witzmann, Frank A.; Zhen, Gehua; Wang, Xiao; Cao, Xu; Roodman, G. David; Marks, Andrew R.; Guise, Theresa A.

2015-01-01

Cancer-associated muscle weakness is poorly understood and there is no effective treatment. Here, we find that seven different mouse models of human osteolytic bone metastases, representing breast, lung and prostate cancers, as well as multiple myeloma exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that TGF-β, released from the bone surface as a result of metastasis-induced bone destruction upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor/calcium (Ca2+) release channel (RyR1). The oxidized RyR1 channels leaked Ca2+, resulting in lower intracellular signaling required for proper muscle contraction. We found that inhibiting RyR1 leak, TGF-β signaling, TGF-β release from bone or Nox4 all improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast cancer- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, higher levels of Nox4 protein and Nox4 binding to RyR1, and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a non-malignant metabolic bone disorder associated with increased TGF-β activity. Thus, metastasis-induced TGF-β release from bone contributes to muscle weakness by decreasing Ca2+-induced muscle force production. PMID:26457758

Protein oxidation and proteolysis during storage and in vitro digestion of pork and beef patties.

PubMed

Rysman, Tine; Van Hecke, Thomas; Van Poucke, Christof; De Smet, Stefaan; Van Royen, Geert

2016-10-15

The effect of protein oxidation on proteolysis during meat digestion was investigated following storage and subsequent in vitro digestion of beef and pork patties. Protein oxidation was evaluated as thiol oxidation, total carbonylation, and specific carbonylation (α-amino adipic and γ-glutamic semialdehyde). Furthermore, 4-hydroxyphenylalanine, a hydroxylation product of phenylalanine, was identified and quantified as a new protein oxidation marker. After 7days of chilled illuminated storage (4°C), significant oxidative modifications were quantified and the oxidative degradation was continued during in vitro digestion. The observed effects were more abundant in beef patties. Protein oxidation before digestion resulted in impaired proteolysis during digestion. Copyright © 2016 Elsevier Ltd. All rights reserved.

Oxidative Stress and Autophagy in Cardiovascular Homeostasis

PubMed Central

Morales, Cyndi R.; Pedrozo, Zully; Lavandero, Sergio

2014-01-01

Abstract Significance: Autophagy is an evolutionarily ancient process of intracellular protein and organelle recycling required to maintain cellular homeostasis in the face of a wide variety of stresses. Dysregulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) leads to oxidative damage. Both autophagy and ROS/RNS serve pathological or adaptive roles within cardiomyocytes, depending on the context. Recent Advances: ROS/RNS and autophagy communicate with each other via both transcriptional and post-translational events. This cross talk, in turn, regulates the structural integrity of cardiomyocytes, promotes proteostasis, and reduces inflammation, events critical to disease pathogenesis. Critical Issues: Dysregulation of either autophagy or redox state has been implicated in many cardiovascular diseases. Cardiomyocytes are rich in mitochondria, which make them particularly sensitive to oxidative damage. Maintenance of mitochondrial homeostasis and elimination of defective mitochondria are each critical to the maintenance of redox homeostasis. Future Directions: The complex interplay between autophagy and oxidative stress underlies a wide range of physiological and pathological events and its elucidation holds promise of potential clinical applicability. Antioxid. Redox Signal. 20, 507–518. PMID:23641894

Impact of Lipid and Protein Co-oxidation on Digestibility of Dairy Proteins in Oil-in-Water (O/W) Emulsions.

PubMed

Obando, Mónica; Papastergiadis, Antonios; Li, Shanshan; De Meulenaer, Bruno

2015-11-11

Enrichment of polyunsaturated fatty acids (PUFAs) is a growing trend in the food industry. However, PUFAs are known to be susceptible to lipid oxidation. It has been shown that oxidizing lipids react with proteins present in the food and that as a result polymeric protein complexes are produced. Therefore, the aim of this work was to investigate the impact of lipid and protein co-oxidation on protein digestibility. Casein and whey protein (6 mg/mL) based emulsions with 1% oil with different levels of PUFAs were subjected to respectively autoxidation and photo-oxidation. Upon autoxidation at 70 °C, protein digestibility of whey protein based emulsions containing fish oil decreased to 47.7 ± 0.8% after 48 h, whereas in the controls without oil 67.8 ± 0.7% was observed. Upon photo-oxidation at 4 °C during 30 days, mainly casein-based emulsions containing fish oil were affected: the digestibility amounted to 43.9 ± 1.2%, whereas in the control casein solutions without oil, 72.6 ± 0.2% of the proteins were digestible. Emulsions containing oils with high PUFA levels were more prone to lipid oxidation and thus upon progressive oxidation showed a higher impact on protein digestibility.

Retinol Binding Protein 4 in Relation to Diet, Inflammation, Immunity, and Cardiovascular Diseases12

PubMed Central

Zabetian-Targhi, Fateme; Mahmoudi, Mohammad J; Rezaei, Nima; Mahmoudi, Maryam

2015-01-01

Retinol binding protein 4 (RBP4), previously called retinol binding protein (RBP), is considered a specific carrier of retinol in the blood. It is also an adipokine that has been implicated in the pathophysiology of insulin resistance. RBP4 seems to be correlated with cardiometabolic markers in inflammatory chronic diseases, including obesity, type 2 diabetes, metabolic syndrome, and cardiovascular diseases (CVDs). It has recently been suggested that inflammation produced by RBP4 induces insulin resistance and CVD. The clinical relevance of this hypothesis is discussed in this review. Knowledge concerning the association of RBP4 with inflammation markers, oxidative stress, and CVDs as well as concerning the role of diet and antioxidants in decreasing RBP4 concentrations are discussed. Special attention is given to methodologies used in previously published studies and covariates that should be controlled when planning new studies on this adipokine. PMID:26567199

Ascorbate and Apple Phenolics Affect Protein Oxidation in Emulsion-Type Sausages during Storage and in Vitro Digestion.

PubMed

Rysman, Tine; Van Hecke, Thomas; De Smet, Stefaan; Van Royen, Geert

2016-05-25

The effect of sodium ascorbate and apple phenolics on the oxidative stability of emulsion-type sausages during storage and digestion was investigated. Emulsion-type sausages containing 0.05% sodium ascorbate or 3% freeze-dried apple pomace were subjected to chilled illuminated storage and subsequent in vitro digestion. Lipid oxidation was assessed as TBARS, and protein oxidation was evaluated as thiol oxidation, total carbonyls, and γ-glutamic and α-amino adipic semialdehyde. Proteolysis was measured after digestion to evaluate protein digestibility. The results suggest the presence of protein-ascorbate and protein-phenol interactions, which may decrease protein digestibility and may interfere with spectrophotometric methods for measuring oxidation.

Metabolic states following accumulation of intracellular aggregates: implications for neurodegenerative diseases.

PubMed

Vazquez, Alexei

2013-01-01

The formation of intracellular aggregates is a common etiology of several neurodegenerative diseases. Mitochondrial defects and oxidative stress has been pointed as the major mechanistic links between the accumulation of intracellular aggregates and cell death. In this work we propose a "metabolic cell death by overcrowding" as an alternative hypothesis. Using a model of neuron metabolism, we predict that as the concentration of protein aggregates increases the neurons transit through three different metabolic phases. The first phase (0-6 mM) corresponds with the normal neuron state, where the neuronal activity is sustained by the oxidative phosphorylation of lactate. The second phase (6-8.6 mM) is characterized by a mixed utilization of lactate and glucose as energy substrates and a switch from ammonia uptake to ammonia release by neurons. In the third phase (8.6-9.3 mM) neurons are predicted to support their energy demands from glycolysis and an alternative pathway for energy generation, involving reactions from serine synthesis, one carbon metabolism and the glycine cleavage system. The model also predicts a decrease in the maximum neuronal capacity for energy generation with increasing the concentration of protein aggregates. Ultimately this maximum capacity becomes zero when the protein aggregates reach a concentration of about 9.3 mM, predicting the cessation of neuronal activity.

Resveratrol Prevents Ammonia Toxicity in Astroglial Cells

PubMed Central

Guerra, Maria Cristina; Leite, Marina Concli; Souza, Diogo Onofre; Gonçalves, Carlos-Alberto; Gottfried, Carmem

2012-01-01

Ammonia is implicated as a neurotoxin in brain metabolic disorders associated with hyperammonemia. Acute ammonia toxicity can be mediated by an excitotoxic mechanism, oxidative stress and nitric oxide (NO) production. Astrocytes interact with neurons, providing metabolic support and protecting against oxidative stress and excitotoxicity. Astrocytes also convert excess ammonia and glutamate into glutamine via glutamine synthetase (GS). Resveratrol, a polyphenol found in grapes and red wines, exhibits antioxidant and anti-inflammatory properties and modulates glial functions, such as glutamate metabolism. We investigated the effect of resveratrol on the production of reactive oxygen species (ROS), GS activity, S100B secretion, TNF-α, IL-1β and IL-6 levels in astroglial cells exposed to ammonia. Ammonia induced oxidative stress, decreased GS activity and increased cytokines release, probably by a mechanism dependent on protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) pathways. Resveratrol prevented ammonia toxicity by modulating oxidative stress, glial and inflammatory responses. The ERK and nuclear factor-κB (NF-κB) are involved in the protective effect of resveratrol on cytokines proinflammatory release. In contrast, other antioxidants (e.g., ascorbic acid and trolox) were not effective against hyperammonemia. Thus, resveratrol could be used to protect against ammonia-induced neurotoxicity. PMID:23284918

Identification of hydrogen peroxide oxidation sites of alpha A- and alpha B-crystallins.

PubMed

Smith, J B; Jiang, X; Abraham, E C

1997-02-01

The alpha-crystallins are the most abundant structural proteins of the lens and, because of their chaperone activity, contribute to the solubility of the other crystallins. With aging, the lens crystallins undergo a variety of modifications which correlate with a loss of solubility and the development of cataract. A recent study demonstrating that alpha-crystallins exposed in vitro to FeCl3 and H2O2 exhibit decreased chaperone activity, implicates metal catalyzed oxidations of alpha-crystallins in this loss of solubility. The present study has determined that alpha-crystallins incubated with FeCl3 and H2O2 are modified by the nearly complete oxidation of all methionine residues to methionine sulfoxide, with no other detectable reaction products. The modifications were identified from the molecular weights of peptides formed by enzymatic digestion of the alpha-crystallins and located by tandem mass spectrometric analysis of the fragmentation pattern of the mass spectra of the fragments from peptides with oxidized methionine is loss of 64 Da, which corresponds to loss of CH3SOH from the methionine sulfoxide. These fragments are useful in identifying peptides that include oxidized methionine residues.

Reactive Oxygen Species Inactivation of Surfactant Involves Structural and Functional Alterations to Surfactant Proteins SP-B and SP-C

PubMed Central

Rodríguez-Capote, Karina; Manzanares, Dahis; Haines, Thomas; Possmayer, Fred

2006-01-01

Exposing bovine lipid extract surfactant (BLES), a clinical surfactant, to reactive oxygen species arising from hypochlorous acid or the Fenton reaction resulted in an increase in lipid (conjugated dienes, lipid aldehydes) and protein (carbonyls) oxidation products and a reduction in surface activity. Experiments where oxidized phospholipids (PL) were mixed with BLES demonstrated that this addition hampered BLES biophysical activity. However the effects were only moderately greater than with control PL. These results imply a critical role for protein oxidation. BLES oxidation by either method resulted in alterations in surfactant proteins SP-B and SP-C, as evidenced by altered Coomassie blue and silver staining. Western blot analyses showed depressed reactivity with specific antibodies. Oxidized SP-C showed decreased palmitoylation. Reconstitution experiments employing PL, SP-B, and SP-C isolated from control or oxidized BLES demonstrated that protein oxidation was more deleterious than lipid oxidation. Furthermore, addition of control SP-B can improve samples containing oxidized SP-C, but not vice versa. We conclude that surfactant oxidation arising from reactive oxygen species generated by air pollution or leukocytes interferes with surfactant function through oxidation of surfactant PL and proteins, but that protein oxidation, in particular SP-B modification, produces the major deleterious effects. PMID:16443649

Role of macrophage migration inhibitory factor (MIF) in the effects of oxidative stress on human retinal pigment epithelial cells.

PubMed

Ko, Ji-Ae; Sotani, Yasuyuki; Ibrahim, Diah Gemala; Kiuchi, Yoshiaki

2017-10-01

Proliferative vitreoretinopathy (PVR) is the major cause of treatment failure in individuals who undergo surgery for retinal detachment. The epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells contributes to the pathogenesis of PVR. Oxidative stress is thought to play a role in the progression of retinal diseases including PVR. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line. We found that H 2 O 2 induced the contraction of RPE cells in a three-dimensional collagen gel. Analysis of a cytokine array revealed that H 2 O 2 specifically increased the release of macrophage migration inhibitory factor (MIF) from RPE cells. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that H 2 O 2 increased the expression of MIF in RPE cells. Immunoblot and immunofluorescence analyses revealed that H 2 O 2 upregulated the expression of α-SMA and vimentin and downregulated that of ZO-1 and N-cadherin. Consistent with these observations, the transepithelial electrical resistance of cell was reduced by exposure to H 2 O 2 . The effects of oxidative stress on EMT-related and junctional protein expression as well as on transepithelial electrical resistance were inhibited by antibodies to MIF, but they were not mimicked by treatment with recombinant MIF. Finally, analysis with a profiling array for mitogen-activated protein kinase signalling revealed that H 2 O 2 specifically induced the phosphorylation of p38 mitogen-activated protein kinase. Our results thus suggest that MIF may play a role in induction of the EMT and related processes by oxidative stress in RPE cells and that it might thereby contribute to the pathogenesis of PVR. Proliferative vitreoretinopathy is a major complication of rhegmatogenous retinal detachment, and both oxidative stress and induction of the EMT in RPE cells are thought to contribute to the pathogenesis of this condition. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line ARPE19. Our results thus implicate MIF in induction of the EMT and related processes by oxidative stress in RPE cells and the regulated expression of EMT markers. They further suggest that MIF may play an important role in the pathogenesis of PVR. Copyright © 2017 John Wiley & Sons, Ltd.

Mild Oxidation Promotes and Advanced Oxidation Impairs Remodeling of Human High-Density Lipoprotein in vitro

PubMed Central

Gao, Xuan; Jayaraman, Shobini; Gursky, Olga

2008-01-01

SUMMARY High-density lipoproteins (HDL) prevent atherosclerosis by removing cholesterol from macrophages and by exerting anti-oxidant and anti-inflammatory effects. Oxidation is thought to impair HDL functions, yet certain oxidative modifications may be advantageous; thus, mild oxidation reportedly enhances cell cholesterol uptake by HDL whereas extensive oxidation impairs it. To elucidate the underlying energetic and structural basis, we analyzed the effects of copper and hypochlorite (that preferentially oxidize lipids and proteins, respectively) on thermal stability of plasma spherical HDL. Circular dichroism, light scattering, calorimetry, gel electrophoresis and electron microscopy showed that mild oxidation destabilizes HDL and accelerates protein dissociation and lipoprotein fusion, while extensive oxidation inhibits these reactions; this inhibition correlates with massive protein cross-linking and lipolysis. We propose that mild oxidation lowers kinetic barriers for HDL remodeling due to diminished apolipoprotein affinity for lipids resulting from oxidation of methionine and aromatic residues in apolipoproteins A-I and A-II followed by protein cross-linking into dimers and/or trimers. In contrast, advanced oxidation inhibits protein dissociation and HDL fusion due to lipid re-distribution from core to surface upon lipolysis and to massive protein cross-linking. Our results help reconcile the apparent controversy in the studies of oxidized HDL and suggest that mild oxidation may benefit HDL functions. PMID:18190928

Increased 8-hydroxy-2'-deoxyguanosine in plasma and decreased mRNA expression of human 8-oxoguanine DNA glycosylase 1, anti-oxidant enzymes, mitochondrial biogenesis-related proteins and glycolytic enzymes in leucocytes in patients with systemic lupus erythematosus.

PubMed

Lee, H-T; Lin, C-S; Lee, C-S; Tsai, C-Y; Wei, Y-H

2014-04-01

We measured plasma levels of the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) and leucocyte mRNA expression levels of the genes encoding the 8-OHdG repair enzyme human 8-oxoguanine DNA glycosylase 1 (hOGG1), the anti-oxidant enzymes copper/zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase-1 (GPx-1), GPx-4, glutathione reductase (GR) and glutathione synthetase (GS), the mitochondrial biogenesis-related proteins mtDNA-encoded ND 1 polypeptide (ND1), ND6, ATPase 6, mitochondrial transcription factor A (Tfam), nuclear respiratory factor 1(NRF-1), pyruvate dehydrogenase E1 component alpha subunit (PDHA1), pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1) and hypoxia inducible factor-1α (HIF-1α) and the glycolytic enzymes hexokinase-II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase A (LDHa). We analysed their relevance to oxidative damage in 85 systemic lupus erythematosus (SLE) patients, four complicated SLE patients undergoing rituximab treatment and 45 healthy individuals. SLE patients had higher plasma 8-OHdG levels (P < 0·01) but lower leucocyte expression of the genes encoding hOGG1(P < 0·01), anti-oxidant enzymes (P < 0·05), mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) than healthy individuals. The increase in plasma 8-OHdG was correlated positively with the elevation of leucocyte expression of the genes encoding hOGG1 (P < 0·05), anti-oxidant enzymes (P < 0·05), several mitochondrial biogenesis-related proteins (P < 0·05) and glycolytic enzymes (P < 0·05) in lupus patients. The patients, whose leucocyte mtDNA harboured D310 heteroplasmy, exhibited a positive correlation between the mtDNA copy number and expression of ND1, ND6 and ATPase 6 (P < 0·05) and a negative correlation between mtDNA copy number and systemic lupus erythematosus disease activity index (SLEDAI) (P < 0·05), as well as plasma 8-OHdG (P < 0·05). In particular, four complicated SLE patients with increased expression of the genes encoding the anti-oxidant enzymes, GAPDH, Tfam and PDHA1, experienced better therapeutic outcomes after rituximab therapy. In conclusion, higher oxidative damage with suboptimal increases in DNA repair, anti-oxidant capacity, mitochondrial biogenesis and glucose metabolism may be implicated in SLE deterioration, and this impairment might be improved by targeted biological therapy. © 2013 British Society for Immunology.

Effects of diet, packaging, and irradiation on protein oxidation, lipid oxidation, and color of raw broiler thigh meat during refrigerated storage.

PubMed

Xiao, S; Zhang, W G; Lee, E J; Ma, C W; Ahn, D U

2011-06-01

This study was designed to evaluate the effects of dietary treatment, packaging, and irradiation singly or in combination on the oxidative stability of broiler chicken thigh meat. A total of 120 four-week-old chickens were divided into 12 pens (10 birds/pen), and 4 pens of broilers were randomly assigned to a control oxidized diet (5% oxidized oil) or an antioxidant-added diet [500 IU of vitamin E + 200 mg/kg of butylated hydroxyanisole (BHA)] and fed for 2 wk. After slaughter, thigh meats were separated, ground, packaged in either oxygen-permeable or oxygen-impermeable vacuum bags, and irradiated at 0 or 3 kGy. Lipid oxidation (TBA-reactive substances), protein oxidation (carbonyl), and color of the meat were measured at 1, 4, and 7 d of refrigerated storage. The lipid and protein oxidation of thigh meats from birds fed the diet supplemented with antioxidants (vitamin E + BHA) was significantly lower than the lipid and protein oxidation of birds fed the control diet, whereas the lipid and protein oxidation of broilers fed the oxidized oil diet was higher than that of birds fed the control diet. Vacuum packaging slowed, but irradiation accelerated, the lipid and protein oxidation of thigh meat during storage. Dietary antioxidants (vitamin E + BHA) and irradiation treatments showed a stronger effect on lipid oxidation than on protein oxidation. A significant correlation between lipid and protein oxidation in meat was found during storage. Dietary supplementation of vitamin E + BHA and the irradiation treatment increased the lightness and redness of thigh meat, respectively. It is suggested that appropriate use of dietary antioxidants in combination with packaging could be effective in minimizing oxidative changes in irradiated raw chicken thigh meat.

Targeting Heat Shock Proteins in Cancer: A Promising Therapeutic Approach

PubMed Central

Burns, Timothy F.

2017-01-01

Heat shock proteins (HSPs) are a large family of chaperones that are involved in protein folding and maturation of a variety of “client” proteins protecting them from degradation, oxidative stress, hypoxia, and thermal stress. Hence, they are significant regulators of cellular proliferation, differentiation and strongly implicated in the molecular orchestration of cancer development and progression as many of their clients are well established oncoproteins in multiple tumor types. Interestingly, tumor cells are more HSP chaperonage-dependent than normal cells for proliferation and survival because the oncoproteins in cancer cells are often misfolded and require augmented chaperonage activity for correction. This led to the development of several inhibitors of HSP90 and other HSPs that have shown promise both preclinically and clinically in the treatment of cancer. In this article, we comprehensively review the roles of some of the important HSPs in cancer, and how targeting them could be efficacious, especially when traditional cancer therapies fail. PMID:28914774

Docosahexaenoic acid protects from dendritic pathology in an Alzheimer's disease mouse model.

PubMed

Calon, Frédéric; Lim, Giselle P; Yang, Fusheng; Morihara, Takashi; Teter, Bruce; Ubeda, Oliver; Rostaing, Phillippe; Triller, Antoine; Salem, Norman; Ashe, Karen H; Frautschy, Sally A; Cole, Greg M

2004-09-02

Learning and memory depend on dendritic spine actin assembly and docosahexaenoic acid (DHA), an essential n-3 (omega-3) polyunsaturated fatty acid (PFA). High DHA consumption is associated with reduced Alzheimer's disease (AD) risk, yet mechanisms and therapeutic potential remain elusive. Here, we report that reduction of dietary n-3 PFA in an AD mouse model resulted in 80%-90% losses of the p85alpha subunit of phosphatidylinositol 3-kinase and the postsynaptic actin-regulating protein drebrin, as in AD brain. The loss of postsynaptic proteins was associated with increased oxidation, without concomitant neuron or presynaptic protein loss. n-3 PFA depletion increased caspase-cleaved actin, which was localized in dendrites ultrastructurally. Treatment of n-3 PFA-restricted mice with DHA protected against these effects and behavioral deficits and increased antiapoptotic BAD phosphorylation. Since n-3 PFAs are essential for p85-mediated CNS insulin signaling and selective protection of postsynaptic proteins, these findings have implications for neurodegenerative diseases where synaptic loss is critical, especially AD.

Mitochondrial protein hyperacetylation in the failing heart

PubMed Central

Horton, Julie L.; Martin, Ola J.; Lai, Ling; Richards, Alicia L.; Vega, Rick B.; Leone, Teresa C.; Pagliarini, David J.; Muoio, Deborah M.; Bedi, Kenneth C.; Coon, Joshua J.

2016-01-01

Myocardial fuel and energy metabolic derangements contribute to the pathogenesis of heart failure. Recent evidence implicates posttranslational mechanisms in the energy metabolic disturbances that contribute to the pathogenesis of heart failure. We hypothesized that accumulation of metabolite intermediates of fuel oxidation pathways drives posttranslational modifications of mitochondrial proteins during the development of heart failure. Myocardial acetylproteomics demonstrated extensive mitochondrial protein lysine hyperacetylation in the early stages of heart failure in well-defined mouse models and the in end-stage failing human heart. To determine the functional impact of increased mitochondrial protein acetylation, we focused on succinate dehydrogenase A (SDHA), a critical component of both the tricarboxylic acid (TCA) cycle and respiratory complex II. An acetyl-mimetic mutation targeting an SDHA lysine residue shown to be hyperacetylated in the failing human heart reduced catalytic function and reduced complex II–driven respiration. These results identify alterations in mitochondrial acetyl-CoA homeostasis as a potential driver of the development of energy metabolic derangements that contribute to heart failure. PMID:26998524

Mitochondrial protein hyperacetylation in the failing heart.

PubMed

Horton, Julie L; Martin, Ola J; Lai, Ling; Riley, Nicholas M; Richards, Alicia L; Vega, Rick B; Leone, Teresa C; Pagliarini, David J; Muoio, Deborah M; Bedi, Kenneth C; Margulies, Kenneth B; Coon, Joshua J; Kelly, Daniel P

2016-02-01

Myocardial fuel and energy metabolic derangements contribute to the pathogenesis of heart failure. Recent evidence implicates posttranslational mechanisms in the energy metabolic disturbances that contribute to the pathogenesis of heart failure. We hypothesized that accumulation of metabolite intermediates of fuel oxidation pathways drives posttranslational modifications of mitochondrial proteins during the development of heart failure. Myocardial acetylproteomics demonstrated extensive mitochondrial protein lysine hyperacetylation in the early stages of heart failure in well-defined mouse models and the in end-stage failing human heart. To determine the functional impact of increased mitochondrial protein acetylation, we focused on succinate dehydrogenase A (SDHA), a critical component of both the tricarboxylic acid (TCA) cycle and respiratory complex II. An acetyl-mimetic mutation targeting an SDHA lysine residue shown to be hyperacetylated in the failing human heart reduced catalytic function and reduced complex II-driven respiration. These results identify alterations in mitochondrial acetyl-CoA homeostasis as a potential driver of the development of energy metabolic derangements that contribute to heart failure.

Agmatine induces Nrf2 and protects against corticosterone effects in hippocampal neuronal cell line.

PubMed

Freitas, Andiara E; Egea, Javier; Buendía, Izaskun; Navarro, Elisa; Rada, Patricia; Cuadrado, Antonio; Rodrigues, Ana Lúcia S; López, Manuela G

2015-01-01

Hyperactivation of the hypothalamic-pituitary-adrenal axis is a common finding in major depression; this may lead to increased levels of cortisol, which are known to cause oxidative stress imbalance and apoptotic neuronal cell death, particularly in the hippocampus, a key region implicated in mood regulation. Agmatine, an endogenous metabolite of L-arginine, has been proposed for the treatment of major depression. Corticosterone induced apoptotic cell death and increased ROS production in cultured hippocampal neuronal cells, effects that were abolished in a concentration- and time-dependent manner by agmatine. Interestingly, the combination of sub-effective concentrations of agmatine with fluoxetine or imipramine afforded synergic protection. The neuroprotective effect of agmatine was abolished by yohimbine (α2-adrenoceptor antagonist), ketanserin (5-HT2A receptor antagonist), LY294002 (PI3K inhibitor), PD98059 (MEK1/2 inhibitor), SnPP (HO-1 inhibitor), and cycloheximide (protein synthesis inhibitor). Agmatine increased Akt and ERK phosphorylation and induced the transcription factor Nrf2 and the proteins HO-1 and GCLc; induction of these proteins was prevented by yohimbine, ketanserin, LY294002, and PD98059. In conclusion, agmatine affords neuroprotection against corticosterone effects by a mechanism that implicates Nrf2 induction via α2-adrenergic and 5-HT2A receptors, Akt and ERK pathways, and HO-1 and GCLc expression.

Honest sexual signalling mediated by parasite and testosterone effects on oxidative balance.

PubMed

Mougeot, Francois; Martínez-Padilla, Jesús; Webster, Lucy M I; Blount, Jonathan D; Pérez-Rodríguez, Lorenzo; Piertney, Stuart B

2009-03-22

Extravagant ornaments evolved to advertise their bearers' quality, the honesty of the signal being ensured by the cost paid to produce or maintain it. The oxidation handicap hypothesis (OHH) proposes that a main cost of testosterone-dependent ornamentation is oxidative stress, a condition whereby the production of reactive oxygen and nitrogen species (ROS/RNS) overwhelms the capacity of antioxidant defences. ROS/RNS are unstable, very reactive by-products of normal metabolic processes that can cause extensive damage to key biomolecules (cellular proteins, lipids and DNA). Oxidative stress has been implicated in the aetiology of many diseases and could link ornamentation and genetic variation in fitness-related traits. We tested the OHH in a free-living bird, the red grouse. We show that elevated testosterone enhanced ornamentation and increased circulating antioxidant levels, but caused oxidative damage. Males with smaller ornaments suffered more oxidative damage than those with larger ornaments when forced to increase testosterone levels, consistent with a handicap mechanism. Parasites depleted antioxidant defences, caused oxidative damage and reduced ornament expression. Oxidative damage extent and the ability of males to increase antioxidant defences also explained the impacts of testosterone and parasites on ornamentation within treatment groups. Because oxidative stress is intimately linked to immune function, parasite resistance and fitness, it provides a reliable currency in the trade-off between individual health and ornamentation. The costs induced by oxidative stress can apply to a wide range of signals, which are testosterone-dependent or coloured by pigments with antioxidant properties.

Honest sexual signalling mediated by parasite and testosterone effects on oxidative balance

PubMed Central

Mougeot, Francois; Martínez-Padilla, Jesu´s; Webster, Lucy M.I.; Blount, Jonathan D.; Pérez-Rodríguez, Lorenzo; Piertney, Stuart B.

2008-01-01

Extravagant ornaments evolved to advertise their bearers' quality, the honesty of the signal being ensured by the cost paid to produce or maintain it. The oxidation handicap hypothesis (OHH) proposes that a main cost of testosterone-dependent ornamentation is oxidative stress, a condition whereby the production of reactive oxygen and nitrogen species (ROS/RNS) overwhelms the capacity of antioxidant defences. ROS/RNS are unstable, very reactive by-products of normal metabolic processes that can cause extensive damage to key biomolecules (cellular proteins, lipids and DNA). Oxidative stress has been implicated in the aetiology of many diseases and could link ornamentation and genetic variation in fitness-related traits. We tested the OHH in a free-living bird, the red grouse. We show that elevated testosterone enhanced ornamentation and increased circulating antioxidant levels, but caused oxidative damage. Males with smaller ornaments suffered more oxidative damage than those with larger ornaments when forced to increase testosterone levels, consistent with a handicap mechanism. Parasites depleted antioxidant defences, caused oxidative damage and reduced ornament expression. Oxidative damage extent and the ability of males to increase antioxidant defences also explained the impacts of testosterone and parasites on ornamentation within treatment groups. Because oxidative stress is intimately linked to immune function, parasite resistance and fitness, it provides a reliable currency in the trade-off between individual health and ornamentation. The costs induced by oxidative stress can apply to a wide range of signals, which are testosterone-dependent or coloured by pigments with antioxidant properties. PMID:19129122

A PerR-like protein involved in response to oxidative stress in the extreme bacterium Deinococcus radiodurans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Chengzhi; Wang, Liangyan; Li, Tao

2014-07-18

Highlights: • We report a novel PerR-like protein of Fur family in D. radiodurans that is not annotated in the current database. • drperR responses to H{sub 2}O{sub 2} and functions as a negative regulator of katE and dps. • We provided implications on how to utilize sequenced genome data and the importance of genome data mining. • This study adds knowledge to complicated regulatory network that responds to ROS stress in D. radiodurans. - Abstract: Response and defense systems against reactive oxygen species (ROS) contribute to the remarkable resistance of Deinococcus radiodurans to oxidative stress induced by oxidants ormore » radiation. However, mechanisms involved in ROS response and defense systems of D. radiodurans are not well understood. Fur family proteins are important in ROS response. Only a single Fur homolog is predicted by sequence similarity in the current D. radiodurans genome database. Our bioinformatics analysis demonstrated an additional guanine nucleotide in the genome of D. radiodurans that is not in the database, leading to the discovery of another Fur homolog DrPerR. Gene disruption mutant of DrPerR showed enhanced resistance to hydrogen peroxide (H{sub 2}O{sub 2}) and increased catalase activity in cell extracts. Real-time PCR results indicated that DrPerR functions as a repressor of the catalase gene katE. Meanwhile, derepression of dps (DNA-binding proteins from starved cells) gene under H{sub 2}O{sub 2} stress by DrPerR point to its regulatory role in metal ions hemostasis. Thus, DrPerR might function as a Fur homolog protein which is involved in ROS response and defense. These results help clarify the complicated regulatory network that responds to ROS stress in D. radiodurans.« less

Diet-induced obesity associated with steatosis, oxidative stress, and inflammation in liver.

PubMed

Peng, Yanhua; Rideout, Drew; Rakita, Steven; Lee, James; Murr, Michel

2012-01-01

Obesity induces steatosis and increases oxidative stress, as well as chronic inflammation in the liver. The balance between lipogenesis and lipolysis is disrupted in obese animals. At a cellular level, the changes in metabolic sensors and energy regulators are poorly understood. We hypothesized that diet-induced steatosis increases oxidative stress, inflammation, and changes the metabolic regulators to promote energy storage in mice. The setting was a university-affiliated basic science research laboratory. Four-week-old C57BL mice were fed a high-fat diet (n = 8) or regular chow (n = 8) for 7 weeks. The liver sections were stained for fat content and immunofluorescence. Liver homogenates were used for protein analysis by immunoblotting and mRNA analysis by reverse transcriptase-polymerase chain reaction. The gels were quantified using densitometry P ≤ .05 was considered significant. The high-fat diet upregulated protein kinase-C atypical isoforms ζ and λ and decreased glucose tolerance and the interaction of insulin receptor substrate 2 with phosphoinositide kinase-3. The high-fat diet increased the transcriptional factors liver X receptor (4321 ± 98 versus 2981 ± 80) and carbohydrate response element-binding protein (5132 ± 135 versus 3076 ± 91), the lipogenesis genes fatty acid binding protein 5, stearoyl-co-enzyme A desaturase-1, and acetyl-co-enzyme A carboxylase protein, and fatty acid synthesis. The high-fat diet decreased 5'-adenosine monophosphate-activated protein kinase (2561 ± 78 versus 1765 ± 65), glucokinase-3β (2.214 ± 34 versus 3356 ± 86), and SIRT1 (2015 ± 76 versus 3567 ± 104) and increased tumor necrosis factor-α (3415 ± 112 versus 2042 ± 65), nuclear factor kappa B (5123 ± 201 versus 2562 ± 103), cyclooxygenase-2 (4230 ± 113 versus 2473 ± 98), nicotinamide-adenine dinucleotide phosphate oxidase (3501 ± 106 versus 1600 ± 69) and reactive oxygen species production (all P < .001, obese mice versus lean mice). A high-fat diet impairs glucose tolerance and hepatic insulin signaling, upregulates transcriptional and translational activities that promote lipogenesis, cytokine production, proinflammatory signaling, and oxidative stress, and downregulates lipolysis. Understanding the complex cellular signals triggered by obesity might have profound clinical implications. Published by Elsevier Inc.

Redox proteomic analysis of serum from aortic anerurysm patients: insights on oxidation of specific protein target.

PubMed

Spadaccio, Cristiano; Coccia, Raffaella; Perluigi, Marzia; Pupo, Gilda; Schininà, Maria Eugenia; Giorgi, Alessandra; Blarzino, Carla; Nappi, Francesco; Sutherland, Fraser W; Chello, Massimo; Di Domenico, Fabio

2016-06-21

oxidative stress is undoubtedly one of the main players in abdominal aortic aneurysm (AAA) pathophysiology. Recent studies in AAA patients reported an increase in the indices of oxidative damage at the tissue level and in biological fluids coupled with the loss of counter-regulatory mechanisms of protection from oxidative stress. We recently reported, in a proteomic analysis of AAA patient sera, changes in the expression of several proteins exerting important modulatory activities on cellular proliferation, differentiation and response to damage. This study aimed to explore the involvement of protein oxidation, at peripheral levels, in AAA. a redox proteomic approach was used to investigate total and specific protein carbonylation and protein-bound 4-hydroxy-2-nonenal (HNE) in the serum of AAA patients compared with age-matched controls. our results show increased oxidative damage to protein as indexed by the total carbonyl levels and total protein-bound HNE. By redox proteomics we identified specific carbonylation of three serum proteins: serum retinol-binding protein, vitamin D-binding protein and fibrinogen α-chain HNE. We also identified increased protein-bound HNE levels for hemopexin, IgK chain C region and IgK chain V-III region SIE. In addition we found a high correlation between specific protein carbonylation and protein-bound HNE and the aortic diameter. Moreover the analysis of serum proteins with antioxidant activity demonstrates the oxidation of albumin together with the overexpression of transferrin, haptoglobin and HSPs 90, 70, 60 and 32. this study support the involvement of oxidative stress in the pathogenesis of AAA and might provide a further degree of knowledge in the cause-effect role of oxidative stress shedding new light on the molecular candidates involved in the disease.

Exercise through a cardiac rehabilitation program attenuates oxidative stress in patients submitted to coronary artery bypass grafting.

PubMed

Taty Zau, José Francisco; Costa Zeferino, Rodrigo; Sandrine Mota, Nádia; Fernandes Martins, Gerez; Manoel Serra, Salvador; Bonates da Cunha, Therezil; Medeiros Lima, Daniel; Bragança Pereira, Basilio de; Matos do Nascimento, Emília; Filho, Danilo Wilhelm; Curi Pedrosa, Rozangela; Pedrosa, Roberto Coury

2018-12-01

Cardiovascular disease is the main cause of morbidity and mortality in the world and oxidative stress has been implicated in the pathogenesis. Cardiac rehabilitation in patients with coronary artery disease submitted to coronary artery bypass grafting may prevent cardiovascular events probably through the attenuation of oxidative stress. The aim of this study was to evaluate the benefits of a cardiac rehabilitation program in the control of the systemic oxidative stress. The studied population consisted of 40 patients, with chronic stable coronary artery disease submitted to coronary artery bypass grafting, who attended a cardiac rehabilitation program. Biomarkers of oxidative stress were evaluated in the blood of these patients at different moments. After the onset of cardiac rehabilitation, there was a significant and progressive decrease in thiobarbituric acid reactive substances levels and protein carbonyls, an initial increase and subsequent decrease in superoxide dismutase, catalase and glutathione peroxidase activities. Also, a progressive increase of uric acid, while ferric reducing antioxidant power levels increased only at the end of the cardiac rehabilitation and a tendency to increase of glutathione contents. The results suggest that regular exercise through a cardiac rehabilitation program can attenuate oxidative stress in chronic coronary artery disease patients submitted to coronary artery bypass grafting.

Pharmacological models and approaches for pathophysiological conditions associated with hypoxia and oxidative stress.

PubMed

Farías, Jorge G; Herrera, Emilio A; Carrasco-Pozo, Catalina; Sotomayor-Zárate, Ramón; Cruz, Gonzalo; Morales, Paola; Castillo, Rodrigo L

2016-02-01

Hypoxia is the failure of oxygenation at the tissue level, where the reduced oxygen delivered is not enough to satisfy tissue demands. Metabolic depression is the physiological adaptation associated with reduced oxygen consumption, which evidently does not cause any harm to organs that are exposed to acute and short hypoxic insults. Oxidative stress (OS) refers to the imbalance between the generation of reactive oxygen species (ROS) and the ability of endogenous antioxidant systems to scavenge ROS, where ROS overwhelms the antioxidant capacity. Oxidative stress plays a crucial role in the pathogenesis of diseases related to hypoxia during intrauterine development and postnatal life. Thus, excessive ROS are implicated in the irreversible damage to cell membranes, DNA, and other cellular structures by oxidizing lipids, proteins, and nucleic acids. Here, we describe several pathophysiological conditions and in vivo and ex vivo models developed for the study of hypoxic and oxidative stress injury. We reviewed existing literature on the responses to hypoxia and oxidative stress of the cardiovascular, renal, reproductive, and central nervous systems, and discussed paradigms of chronic and intermittent hypobaric hypoxia. This systematic review is a critical analysis of the advantages in the application of some experimental strategies and their contributions leading to novel pharmacological therapies. Copyright © 2015 Elsevier Inc. All rights reserved.

Detection of Nitric Oxide by the Sample Analysis at Mars (SAM) Instrument Implications for the Presence of Nitrates

NASA Technical Reports Server (NTRS)

Navarro-Gonzalez, R.; Stern, J.; Freissinet, C.; Franz, H. B.; Eigenbrode, J. L..; McKay, C. P.; Coll, P.; Sutter, B.; Archer, D.; McAdam, A.;

Detection of Catalase as a major protein target of the lipid peroxidation product 4-HNE and the lack of its genetic association as a risk factor in SLE

PubMed Central

D'souza, Anil; Kurien, Biji T; Rodgers, Rosalie; Shenoi, Jaideep; Kurono, Sadamu; Matsumoto, Hiroyuki; Hensley, Kenneth; Nath, Swapan K; Scofield, R Hal

2008-01-01

Background Systemic lupus erythematosus (SLE) is a multifactorial disorder characterized by the presence of autoantibodies. We and others have implicated free radical mediated peroxidative damage in the pathogenesis of SLE. Since harmful free radical products are formed during this oxidative process, including 4-hydroxy 2-nonenol (4-HNE) and malondialdehyde (MDA), we hypothesized that specific HNE-protein adducts would be present in SLE red blood cell (RBC) membranes. Catalase is located on chromosome 11p13 where linkage analysis has revealed a marker in the same region of the genome among families with thrombocytopenia, a clinical manifestation associated with severe lupus in SLE affected pedigrees. Moreover, SLE afflicts African-Americans three times more frequently than their European-American counterparts. Hence we investigated the effects of a genetic polymorphism of catalase on risk and severity of SLE in 48 pedigrees with African American ancestry. Methods Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis was used to identify the protein modified by HNE, following Coomassie staining to visualize the bands on the acrylamide gels. Genotyping analysis for the C → T, -262 bp polymorphism in the promoter region of catalase was performed by PCR-RFLP and direct PCR-sequencing. We used a "pedigree disequilibrium test" for the family based association analysis, implemented in the PDT program to analyze the genotyping results. Results We found two proteins to be HNE-modified, migrating around 80 and 50 kD respectively. Tryptic digestion followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the Coomassie stained 80 kD band revealed that the target of HNE modification was catalase, a protein shown to associate with RBC membrane proteins. All the test statistics carried out on the genotyping analysis for the C → T, -262 bp polymorphism in the promoter region of catalase were non-significant (p > 0.05) in our data, which suggested that this SNP is not associated with SLE. Conclusion Our results indicate that catalase is one of the proteins modified due to oxidative stress. However, catalase may not be a susceptibility gene for SLE. Nonetheless, catalase is oxidatively modified among SLE patients. This suggests a possible role between oxidative modification of catalase and its affects on enzymatic activity in SLE. An oxidatively modified catalase could be one of the reasons for lower enzymatic activity among SLE subjects, which in turn could favor the accumulation of deleterious hydrogen peroxide. Furthermore, HNE-products are potential neoantigens and could be involved in the pathogenesis of SLE. Decrease in catalase activity could affect the oxidant-antioxidant balance. Chronic disturbance of this balance in patients with SLE may work favorably for the premature onset of atherogenesis with severe vascular effect. PMID:18606005

Skin beautification with oral non-hydrolized versions of carnosine and carcinine: Effective therapeutic management and cosmetic skincare solutions against oxidative glycation and free-radical production as a causal mechanism of diabetic complications and skin aging.

PubMed

Babizhayev, Mark A; Deyev, Anatoliy I; Savel'yeva, Ekaterina L; Lankin, Vadim Z; Yegorov, Yegor E

2012-10-01

Advanced glycation Maillard reaction end products (AGEs) are causing the complications of diabetes and skin aging, primarily via adventitious and cross-linking of proteins. Long-lived proteins such as structural collagen are particularly implicated as pathogenic targets of AGE processes. The formation of α-dicarbonyl compounds represents an important step for cross-linking proteins in the glycation or Maillard reaction. The purpose of this study was to investigate the contribution of glycation coupled to the glycation free-radical oxidation reactions as markers of protein damage in the aging of skin tissue proteins and diabetes. To elucidate the mechanism for the cross-linking reaction, we studied the reaction between a three-carbon α-dicarbonyl compound, methylglyoxal, and amino acids using EPR spectroscopy, a spectrophotometric kinetic assay of superoxide anion production at the site of glycation and a chemiluminescence technique. The transglycating activity, inhibition of transition metal ions peroxidative catalysts, resistance to hydrolysis of carnosine mimetic peptide-based compounds with carnosinase and the protective effects of carnosine, carcinine and related compounds against the oxidative damage of proteins and lipid membranes were assessed in a number of biochemical and model systems. A 4-month randomized, double-blind, controlled study was undertaken including 42 subjects where the oral supplement of non-hydrolized carnosine (Can-C Plus® formulation) was tested against placebo for 3 months followed by a 1-month supplement-free period for both groups to assess lasting effects. Assessment of the age-related skin parameters and oral treatment efficacy measurements included objective skin surface evaluation with Visioscan® VC 98 and visual assessment of skin appearance parameters. The results together confirm that a direct one-electron transfer between a Schiff base methylglyoxal dialkylimine (or its protonated form) and methylglyoxal is responsible for the generation of the cross-linked radical cation and the radical counteranion of methylglyoxal. Under aerobic conditions, molecular oxygen can then accept an electron from the methylglyoxal anion to generate the superoxide radical anion causing the propagation of oxidative stress chain reactions in the presence of transition metal ions. Carnosine stabilized from enzymatic hydrolysis, carcinine and leucyl-histidylhydrazide in patented formulations thereof, demonstrate the Schiff bases' transglycating activities concomitant with glycation site specific antioxidant activities and protection of proprietary antioxidant enzymes in the skin during aging and with diabetes lesions. During oral supplementation with stabilized from enzymatic hydrolysis carnosine (Can-C Plus® formulation), the skin parameters investigated showed a continuous and significant improvement in the active group during the 3 months of supplementation as compared to placebo. Visual investigation showed improvement of the overall skin appearance and a reduction of fine lines. No treatment-related side effects were reported. The finding that already-formed AGE cross-links can be pharmacologically severed and attendant pathology thereby reversed by non-hydrolized carnosine or carcinine in patented oral formulations thereof has broad implications for the skin beautification and therapeutics of the complications of diabetes and skin diseases associated with aging.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunil, Vasanthi R., E-mail: sunilvr@eohsi.rutgers.edu; Patel-Vayas, Kinal, E-mail: kinalv5@gmail.com; Shen, Jianliang, E-mail: jianliangs@gmail.com

Lung toxicity induced by sulfur mustard is associated with inflammation and oxidative stress. To elucidate mechanisms mediating pulmonary damage, we used 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. Male mice (B6129) were treated intratracheally with CEES (3 or 6 mg/kg) or control. Animals were sacrificed 3, 7 or 14 days later and bronchoalveolar lavage (BAL) fluid and lung tissue collected. Treatment of mice with CEES resulted in an increase in BAL protein, an indication of alveolar epithelial damage, within 3 days. Expression of Ym1, an oxidative stress marker also increased in the lung, along with inducible nitric oxidemore » synthase, and at 14 days, cyclooxygenase-2 and monocyte chemotactic protein-1, inflammatory proteins implicated in tissue injury. These responses were attenuated in mice lacking the p55 receptor for TNF{alpha} (TNFR1-/-), demonstrating that signaling via TNFR1 is key to CEES-induced injury, oxidative stress, and inflammation. CEES-induced upregulation of CuZn-superoxide dismutase (SOD) and MnSOD was delayed or absent in TNFR1-/- mice, relative to WT mice, suggesting that TNF{alpha} mediates early antioxidant responses to lung toxicants. Treatment of WT mice with CEES also resulted in functional alterations in the lung including decreases in compliance and increases in elastance. Additionally, methacholine-induced alterations in total lung resistance and central airway resistance were dampened by CEES. Loss of TNFR1 resulted in blunted functional responses to CEES. These effects were most notable in the airways. These data suggest that targeting TNF{alpha} signaling may be useful in mitigating lung injury, inflammation and functional alterations induced by vesicants.« less

Tacrine is implicated in oxidative stress in the laboratory guinea pig model.

PubMed

Kracmarova, Alzbeta; Bandouchova, Hana; Pikula, Jiri; Pohanka, Miroslav

2012-01-01

Tacrine was the first acetylcholinesterase inhibitor approved for the treatment of Alzheimer disease. The compound is not available for therapeutic purposes as it was withdrawn due to hepatotoxicity of its metabolites. The hepatotoxicity can be decreased by alternative ways of drug administration avoiding thus the first pass effect. The present study is aimed to investigate the influence of intramuscularly administrated tacrine on oxidative stress. Laboratory guinea pigs were exposed to tacrine at doses of 0-800 μg/kg. The animals were euthanized 1 and 24 hours after the exposure. Parameters such as ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS), carbonylated proteins, caspase 3 activity, superoxide dismutase activity and glutathione reductase activity were assessed in the frontal, temporal and occipital lobe, cerebellum, liver, spleen, heart, and kidney. Moreover, levels of glucose, total and HDL cholesterol forms, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, albumin and activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase were assessed in plasma samples. Activities of the enzymatic markers, level of carbonylated proteins in organs and levels of biochemical markers in plasma were only slightly influenced by tacrine. Dose-dependent elevation of the FRAP value was recognized in the brain tissues and the liver. The TBARS value was increased in the kidney and heart 1 and 24 hours, respectively, after exposure. In the study, the effect of tacrine on markers of oxidative stress was proved. Possible positive effects of tacrine on the antioxidant defence in the brain tissue were discussed.

Ion-current-based Proteomic Profiling of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome*

PubMed Central

Tu, Chengjian; Li, Jun; Jiang, Xiaosheng; Sheflin, Lowell G.; Pfeffer, Bruce A.; Behringer, Matthew; Fliesler, Steven J.; Qu, Jun

2013-01-01

Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age- and sex-matched control rats (n = 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ∼40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ion-current data (e.g. signal-to-noise ratio ≥ 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS. PMID:23979708

An Efficient and Rapid Method to Monitor the Oxidative Degradation of Protein Pharmaceuticals: Probing Tyrosine Oxidation with Fluorogenic Derivatization.

PubMed

Bommana, Rupesh; Mozziconacci, Olivier; John Wang, Y; Schöneich, Christian

2017-07-01

The loss of potency of protein therapeutics can be linked to the oxidation of specific amino acid residues leading to a great variety of oxidative modifications. The comprehensive identification of these oxidative modifications requires high-resolution mass spectrometry analysis, which requires time and expensive resources. Here, we propose a fluorogenic derivatization method of oxidized Tyr and Phe yielding benzoxazole derivatives, as an orthogonal technique for the rapid screening of protein oxidation. Four model proteins, IgG1, human growth hormone (hGH), insulin and bovine serum albumin (BSA) were exposed to oxidation via peroxyl radicals and metal-catalyzed reactions and efficiently screened by fluorogenic derivatization of Tyr and Phe oxidation products. Complementary LC-MS analysis was done to identify the extent of methionine oxidation in oxidized proteins. The Fluorogenic derivatization technique can easily be adapted to a 96-well plate, in which several protein formulations can be screened in short time. Representatively for hGH, we show that the formation of benzoxazole parallels the oxidation of Met to methionine sulfoxide which enables estimation of Met oxidation by just recording the fluorescence. Our rapid fluorescence based screening allows for the fast comparison of the stability of multiple formulations.

Nitrite promotes protein carbonylation and Strecker aldehyde formation in experimental fermented sausages: are both events connected?

PubMed

Villaverde, A; Ventanas, J; Estévez, M

2014-12-01

The role played by curing agents (nitrite, ascorbate) on protein oxidation and Strecker aldehyde formation is studied. To fulfill this objective, increasing concentrations of nitrite (0, 75 and 150ppm) and ascorbate (0, 250 and 500ppm) were added to sausages subjected to a 54day drying process. The concurrence of intense proteolysis, protein carbonylation and formation of Strecker aldehydes during processing of sausages suggests that α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) may be implicated in the formation of Strecker aldehydes. The fact that nitrite (150ppm, ingoing amount) significantly promoted the formation of protein carbonyls at early stages of processing and the subsequent formation of Strecker aldehydes provides strength to this hypothesis. Ascorbate (125 and 250ppm) controlled the overall extent of protein carbonylation in sausages without declining the formation of Strecker aldehydes. These results may contribute to understanding the chemistry fundamentals of the positive influence of nitrite on the flavor and overall acceptability of cured muscle foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hydroxynonenal-generated crosslinking fluorophore accumulation in Alzheimer disease reveals a dichotomy of protein turnover.

PubMed

Zhu, Xiongwei; Castellani, Rudy J; Moreira, Paula I; Aliev, Gjumrakch; Shenk, Justin C; Siedlak, Sandra L; Harris, Peggy L R; Fujioka, Hisashi; Sayre, Lawrence M; Szweda, Pamela A; Szweda, Luke I; Smith, Mark A; Perry, George

2012-02-01

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90.

PubMed Central

Conconi, M; Petropoulos, I; Emod, I; Turlin, E; Biville, F; Friguet, B

1998-01-01

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP. PMID:9657982

Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90.

PubMed

Conconi, M; Petropoulos, I; Emod, I; Turlin, E; Biville, F; Friguet, B

1998-07-15

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.

Nuclear factor erythroid-2-related factor regulates LRWD1 expression and cellular adaptation to oxidative stress in human embryonal carcinoma cells.

PubMed

Hung, Jui-Hsiang; Wee, Shi-Kae; Omar, Hany A; Su, Chia-Hui; Chen, Hsing-Yi; Chen, Pin-Shern; Chiu, Chien-Chih; Wu, Ming-Syuan; Teng, Yen-Ni

2018-05-01

Leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1) is implicated in the regulation of signal transduction, transcription, RNA processing and tumor development. However, LRWD1 transcriptional regulation is not fully understood. This study aimed to investigate the relationship between LRWD1 expression and reactive oxygen species (ROS) level in human embryonal carcinoma cell line, NT2/D1 cells, which will help in understanding the transcriptional regulatory role of ROS in cells. Results showed that the exposure of NT2/D1 cells to various concentrations of hydrogen peroxide (H 2 O 2 ) and the nitric oxide (NO) donor sodium nitroprusside (SNP) caused a significant increase in the mRNA and protein expression of LRWD1. In addition, LRWD1 promoter luciferase reporter assay, and Chromatin Immunoprecipitation assay (CHIP assay) showed that nuclear factor erythroid-2-related factor (Nrf2) was involved in the regulation of LRWD1 expression in response to oxidative stress. The involvement of Nrf2 was confirmed by shRNA-mediated knockdown of Nrf2 in NT2/D1 cells, which caused a significant decrease in LRWD1 expression in response to oxidative stress. Similarly, LRWD1 knockdown resulted in the accumulation of H 2 O 2 and superoxide anion radical (O2-). Blocking ROS production by N-acetyl cysteine (NAC) protected NT2/D1 shLRWD1cells from H 2 O 2 -induced cell death. Collectively, oxidative stress increased LRWD1 expression through a Nrf2-dependent mechanism, which plays an important role in cellular adaptation to oxidative stress. These results highlight an evidence, on the molecular level, about LRWD1 transcriptional regulation under oxidative stress. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Tripping Up Trp: Modification of Protein Tryptophan Residues by Reactive Oxygen Species, Modes of Detection, and Biological Consequences

PubMed Central

Ehrenshaft, Marilyn; Deterding, Leesa J.; Mason, Ronald P.

2015-01-01

Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone, and/or polymerization of monomers into dimers, multimers and sometimes into insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions which have only a modest effect on protein size but can have widespread consequences for protein function. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion limited rate with hydroxyl radical, and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral while others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and weaknesses of some of the techniques used to detect these oxidative interactions and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants and microbes. PMID:26393422

Proteomic analysis of endothelial cold-adaptation

PubMed Central

2011-01-01

Background Understanding how human cells in tissue culture adapt to hypothermia may aid in developing new clinical procedures for improved ischemic and hypothermic protection. Human coronary artery endothelial cells grown to confluence at 37°C and then transferred to 25°C become resistant over time to oxidative stress and injury induced by 0°C storage and rewarming. This protection correlates with an increase in intracellular glutathione at 25°C. To help understand the molecular basis of endothelial cold-adaptation, isolated proteins from cold-adapted (25°C/72 h) and pre-adapted cells were analyzed by quantitative proteomic methods and differentially expressed proteins were categorized using the DAVID Bioinformatics Resource. Results Cells adapted to 25°C expressed changes in the abundance of 219 unique proteins representing a broad range of categories such as translation, glycolysis, biosynthetic (anabolic) processes, NAD, cytoskeletal organization, RNA processing, oxidoreductase activity, response-to-stress and cell redox homeostasis. The number of proteins that decreased significantly with cold-adaptation exceeded the number that increased by 2:1. Almost half of the decreases were associated with protein metabolic processes and a third were related to anabolic processes including protein, DNA and fatty acid synthesis. Changes consistent with the suppression of cytoskeletal dynamics provided further evidence that cold-adapted cells are in an energy conserving state. Among the specific changes were increases in the abundance and activity of redox proteins glutathione S-transferase, thioredoxin and thioredoxin reductase, which correlated with a decrease in oxidative stress, an increase in protein glutathionylation, and a recovery of reduced protein thiols during rewarming from 0°C. Increases in S-adenosylhomocysteine hydrolase and nicotinamide phosphoribosyltransferase implicate a central role for the methionine-cysteine transulfuration pathway in increasing glutathione levels and the NAD salvage pathway in increasing the reducing capacity of cold-adapted cells. Conclusions Endothelial adaptation to mild-moderate hypothermia down-regulates anabolic processes and increases the reducing capacity of cells to enhance their resistance to oxidation and injury associated with 0°C storage and rewarming. Inducing these characteristics in a clinical setting could potentially limit the damaging effects of energy insufficiency due to ischemia and prevent the disruption of integrated metabolism at low temperatures. PMID:22192797

Apple phenolics as inhibitors of the carbonylation pathway during in vitro metal-catalyzed oxidation of myofibrillar proteins.

PubMed

Rysman, Tine; Utrera, Mariana; Morcuende, David; Van Royen, Geert; Van Weyenberg, Stephanie; De Smet, Stefaan; Estévez, Mario

2016-11-15

The effect of apple phenolics on the oxidative damage caused to myofibrillar proteins by an in vitro metal-catalyzed oxidation system was investigated. Three pure phenolic compounds (chlorogenic acid, (-)-epicatechin and phloridzin) and an apple peel extract were added to myofibrillar proteins in three concentrations (50, 100 and 200μM), and a blank treatment was included as a control. All suspensions were subjected to Fe(3+)/H2O2 oxidation at 37°C during 10days, and protein oxidation was evaluated as carbonylation (α-amino adipic and γ-glutamic semialdehydes) and Schiff base cross-links. Significant inhibition by apple phenolics was found as compared to the control treatment, with (-)-epicatechin being the most efficient antioxidant and phloridzin showing the weakest antioxidant effect. The higher concentrations of apple extract showed effective antioxidant activity against protein oxidation in myofibrillar proteins, emphasizing the potential of apple by-products as natural inhibitors of protein oxidation in meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Oxidative Stress and Antioxidants in the Diagnosis and Therapy of Periodontitis

PubMed Central

Tóthová, L'ubomíra; Celec, Peter

2017-01-01

Oxidative stress has been implicated in the pathogenesis of numerous diseases. However, large interventional studies with antioxidants failed to show benefits in the prevention or treatment of cardiovascular diseases, cancer, or diabetes mellitus. Numerous clinical studies have confirmed the association of oxidative stress markers and periodontitis. Technical and biological variability is high for most of the analyzed markers and none of them seems to be optimal for routine clinical use. In a research setting, analysis of a palette of oxidative stress markers is needed to cover lipid peroxidation, protein oxidation, and the antioxidant status. The source of reactive oxygen species and their role in the pathogenesis of periodontitis remains unclear. Interventional experiments indicate that oxidative stress might be more than just a simple consequence of the inflammation. Small studies have confirmed that some antioxidants could have therapeutic value at least as an addition to the standard non-surgical treatment of periodontitis. A clear evidence for the efficiency of antioxidant treatment in large patient cohorts is lacking. Potentially, because lowering of oxidative stress markers might be a secondary effect of anti-inflammatory or antibacterial agents. As the field of research of oxidative stress in periodontitis gains attraction and the number of relevant published papers is increasing a systematic overview of the conducted observational and interventional studies is needed. This review summarizes the currently available literature linking oxidative stress and periodontitis and points toward the potential of adjuvant antioxidant treatment, especially in cases where standard treatment fails to improve the periodontal status. PMID:29311982

Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease.

PubMed

Ahmad, Faraz; Singh, Kunal; Das, Debajyoti; Gowaikar, Ruturaj; Shaw, Eisha; Ramachandran, Arathy; Rupanagudi, Khader Valli; Kommaddi, Reddy Peera; Bennett, David A; Ravindranath, Vijayalakshmi

2017-12-01

Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by β-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD. Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APP Swe /PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9-10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains. Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity. We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269-1280.

Curcumin/turmeric solubilized in sodium hydroxide inhibits HNE protein modification--an in vitro study.

PubMed

Kurien, Biji T; Scofield, R Hal

2007-03-21

Free radical mediated lipid peroxidation has been implicated in multiple diseases. A major oxidation by-product of this deleterious process is 4-hydroxy-2-nonenal (HNE). HNE is cytotoxic, mutagenic and genotoxic and is involved in disease pathogenesis. Curcumin, a non-steroidal anti-inflammatory agent (occurring as the yellow pigment found in the rhizomes of the perennial herb Curcuma longa known as turmeric), has emerged as the newest "nutraceutical" agent that has been shown to be efficacious against colon cancer and other disorders, including correcting cystic fibrosis defects. Since curcumin has been reported to have anti-oxidant properties we hypothesized that it will inhibit HNE-modification of a protein substrate. Using an ELISA that employed HNE-modification of solid phase antigen following immobilization, we found that the curcumin solubilized in dilute alkali (5mM sodium hydroxide, pH 11) inhibited HNE-protein modification by 65%. Turmeric also inhibited HNE-protein modification similarly (65%) but at a much lower alkali level (130muM sodium hydroxide, pH 7.6). Alkali by itself (5mM sodium hydroxide, pH 11) was found to enhance HNE modification by as much as 267%. Curcumin/turmeric has to inhibit this alkali enhanced HNE-modification prior to inhibiting the normal HNE protein modification induced by HNE. Thus, inhibition of HNE-modification could be a mechanism by which curcumin exerts its antioxidant effects. The pH at which the inhibition of HNE modification of substrate was observed was close to the physiological pH, making this formulation of curcumin potentially useful practically.

Maternal ethanol consumption reduces Se antioxidant function in placenta and liver of embryos and breastfeeding pups.

PubMed

Nogales, Fátima; Ojeda, M Luisa; Jotty, Karick; Murillo, M Luisa; Carreras, Olimpia

2017-12-01

The fetal alcohol exposition during pregnancy leads to different disorders in offspring, related to the oxidative stress generated by alcohol. It is well-documented that there is an impairment of the antioxidant selenoprotein Glutathione peroxidase (GPx) activity in ethanol offspring during the embryo period, although no-one has described Selenium (Se) status. The aim is to analyze for the first time Se deposits in vivo and Se's biological implication in embryos and placenta after alcohol exposure and in offspring whose mothers continued to drink ethanol during lactation. Se deposits, GPx and glutathione reductase (GR) activity, lipid and protein oxidation and the expression of GPx1 were measured in placenta and liver of both embryos (E-19) and breastfeeding pups (L-21) in control and ethanol groups (20% v/v). Ethanol consumption decreased Se deposits, GPx activity and GPx1 expression, while increasing biomolecular oxidation in placenta and in the liver of E-19 and L-21. The GR/GPx ratio decreased in placenta and in E-19, together with an increase in lipid oxidation, while increased in the liver of L-21 pups with protein oxidation. Ethanol also decreased the GPx1 expression/GPx activity ratio in the liver of E-19 and L-21, indicating that alcohol decreases GPx activity by both depleting Se deposits and promoting GPx inactivation. In placenta GPx activity is proportional to the GPx1 expression found, so the ethanol affects GPx activity in offspring more than in dams. Therefore, Se supplementation therapy in dams could contribute as an interesting antioxidant that prevents fetal alcohol syndrome. Copyright © 2017 Elsevier Inc. All rights reserved.

Redox Proteomics Applied to the Thiol Secretome.

PubMed

Ghezzi, Pietro; Chan, Philippe

2017-03-01

Secreted proteins are important both as signaling molecules and potential biomarkers. Recent Advances: Protein can undergo different types of oxidation, both in physiological conditions or under oxidative stress. Several redox proteomics techniques have been successfully applied to the identification of glutathionylated proteins, an oxidative post-translational modification consisting in the formation of a mixed disulfide between a protein cysteine and glutathione. Redox proteomics has also been used to study other forms of protein oxidation. Because of the highest proportion of free cysteines in the cytosol, redox proteomics of protein thiols has focused, so far, on intracellular proteins. However, plasma proteins, such as transthyretin and albumin, have been described as glutathionylated or cysteinylated. The present review discusses the redox state of protein cysteines in relation to their cellular distribution. We describe the various approaches used to detect secreted glutathionylated proteins, the only thiol modification studied so far in secreted proteins, and the specific problems presented in the study of the secretome. This review focusses on glutathionylated proteins secreted under inflammatory conditions and that may act as soluble mediators (cytokines). Future studies on the redox secretome (including other forms of oxidation) might identify new soluble mediators and biomarkers of oxidative stress. Antioxid. Redox Signal. 26, 299-312.

Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes

PubMed Central

Shi, Liang; Squier, Thomas C; Zachara, John M; Fredrickson, James K

2007-01-01

Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope. PMID:17581116

Effect of alpha-lipoic acid on relieving ammonia stress and hepatic proteomic analyses of broilers.

PubMed

Lu, M; Bai, J; Xu, B; Sun, Q Y; Wei, F X; Tang, X F; Zhang, H F; Li, J; Wang, G L; Yin, Q Q; Li, S Y

2017-01-01

Ammonia in poultry houses not only affects worker health but also induces a variety of poultry diseases. Alpha-lipoic acid (LA) is an effective antioxidant that protects cells against oxidative injury during various toxic and pathological processes. This study was designed to evaluate the mitigating effects of LA supplementation on ammonia stress and hepatic proteome changes in broilers. Male broilers (22 d old) were allocated to 3 groups: (1) a control group without ammonia stress (CTRL); (2) exposure to 70 ppm ammonia (AM); and (3) exposure to 70 ppm ammonia and dietary administration of 300 mg/kg LA (AM+LA). Ammonia exposure significantly decreased broiler growth performance and plasma glutathione peroxidase activity (P < 0.05), and increased plasma malondialdehyde content and glutamic-pyruvic transaminase activity (P < 0.05). These negative effects were eliminated by LA supplementation. Comparative proteomic analyses revealed 291 differentially expressed proteins in the AM group compared to the CTRL and AM+LA groups. A total of 30 proteins were differentially expressed between the AM/CTRL and (AM+LA)/AM groups. The addition of LA restored 24 of these proteins to control levels; these proteins were mainly related to transcription regulation, detoxification, protein translation and degradation, and immune and stress responses. The differentially expressed proteins included the high mobility group box (HMGB) and glutathione S-transferase (GST), which is closely related to immune response and oxidative stress, and collagens, which are implicated in liver injury. The addition of LA to broiler diet may reduce ammonia toxicity by maintaining the antioxidant system, xenobiotic metabolism, and metabolic pathways. © 2016 Poultry Science Association Inc.

Fish proteins as targets of ferrous-catalyzed oxidation: identification of protein carbonyls by fluorescent labeling on two-dimensional gels and MALDI-TOF/TOF mass spectrometry.

PubMed

Pazos, Manuel; da Rocha, Angela Pereira; Roepstorff, Peter; Rogowska-Wrzesinska, Adelina

2011-07-27

Protein oxidation in fish meat is considered to affect negatively the muscle texture. An important source of free radicals taking part in this process is Fenton's reaction dependent on ferrous ions present in the tissue. The aim of this study was to investigate the susceptibility of cod muscle proteins in sarcoplasmic and myofibril fractions to in vitro metal-catalyzed oxidation and to point out protein candidates that might play a major role in the deterioration of fish quality. Extracted control proteins and proteins subjected to free radicals generated by Fe(II)/ascorbate mixture were labeled with fluorescein-5-thiosemicarbazide (FTSC) to tag carbonyl groups and separated by two-dimensional gel electrophoresis. Consecutive visualization of protein carbonyl levels by capturing the FTSC signal and total protein levels by capturing the SyproRuby staining signal allowed us to quantify the relative change in protein carbonyl levels corrected for changes in protein content. Proteins were identified using MALDI-TOF/TOF mass spectrometry and homology-based searches. The results show that freshly extracted cod muscle proteins exhibit a detectable carbonylation background and that the incubation with Fe(II)/ascorbate triggers a further oxidation of both sarcoplasmic and myofibril proteins. Different proteins exhibited various degrees of sensitivity to oxidation processes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), nucleoside diphosphate kinase B (NDK), triosephosphate isomerase, phosphoglycerate mutase, lactate dehydrogenase, creatine kinase, and enolase were the sarcoplasmic proteins most vulnerable to ferrous-catalyzed oxidation. Moreover, NDK, phosphoglycerate mutase, and GAPDH were identified in several spots differing by their pI, and those forms showed different susceptibilities to metal-catalyzed oxidation, indicating that post-translational modifications may change the resistance of proteins to oxidative damage. The Fe(II)/ascorbate treatment significantly increased carbonylation of important structural proteins in fish muscle, mainly actin and myosin, and degradation products of those proteins were observed, some of them exhibiting increased carbonylation levels.

Molecular dynamics simulations of poly (ethylene oxide) hydration and conformation in solutions

NASA Astrophysics Data System (ADS)

Dahal, Udaya; Dormidontova, Elena

Polyethylene oxide (PEO) is one of the most actively used polymers, especially in biomedical applications due to its high hydrophilicity, biocompatibility and potency to inhibit protein adsorption. PEO solubility and conformation in water depends on its capability to form hydrogen bonds. Using atomistic molecular dynamics simulations we investigated the details of water packing around PEO chain and characterized the type and lifetime of hydrogen bonds in aqueous and mixed solvent solutions. The observed polymer chain conformation varies from an extended coil in pure water to collapsed globule in hexane and a helical-like conformation in pure isobutyric acid or isobutyric acid -water mixture in agreement with experimental observations. We'll discuss the implications of protic solvent arrangement and stability of hydrogen bonds on PEO chain conformation and mobility. This research is supported by NSF (DMR-1410928).

Immunohistochemical study of Metallothionein in patients with temporal lobe epilepsy.

PubMed

Juárez-Rebollar, Daniel; Alonso-Vanegas, Mario; Nava-Ruíz, Concepción; Buentello-García, Masao; Yescas-Gómez, Petra; Díaz-Ruíz, Araceli; Rios, Camilo; Méndez-Armenta, Marisela

2017-05-01

Epilepsy is characterized by spontaneous recurrent seizures and temporal lobe epilepsy (TLE) is the most common serious neurological example of acquired and frequent epilepsy. Oxidative stress is recognized as playing a contributing role in several neurological disorders, and most recently have been implicated in acquired epilepsies. The MTs occur in several brain regions and may serve as neuroprotective proteins against reactive oxygen species causing oxidative damage and stress. The main aim of this work was to describe the immunohistochemical localization of MT in the specimens derived from the patients affected by TLE. Histopathological examination showed NeuN, GFAP and MT immunopositive cells that were analyzed for determinate in hippocampal and parietal cortex samples. An increase in the reactive gliosis associated with increased MT expression was observed in patients with TLE. Copyright © 2016 Elsevier Ltd. All rights reserved.

YB-1 Is Important for Late-Stage Embryonic Development, Optimal Cellular Stress Responses, and the Prevention of Premature Senescence

PubMed Central

Lu, Zhi Hong; Books, Jason T.; Ley, Timothy J.

2005-01-01

Proteins containing “cold shock” domains belong to the most evolutionarily conserved family of nucleic acid-binding proteins known among bacteria, plants, and animals. One of these proteins, YB-1, is widely expressed throughout development and has been implicated as a cell survival factor that regulates the transcription and/or translation of many cellular growth and death-related genes. For these reasons, YB-1 deficiency has been predicted to be incompatible with cell survival. However, the majority of YB-1−/− embryos develop normally up to embryonic day 13.5 (E13.5). After E13.5, YB-1−/− embryos exhibit severe growth retardation and progressive mortality, revealing a nonredundant role of YB-1 in late embryonic development. Fibroblasts derived from YB-1−/− embryos displayed a normal rate of protein synthesis and minimal alterations in the transcriptome and proteome but demonstrated reduced abilities to respond to oxidative, genotoxic, and oncogene-induced stresses. YB-1−/− cells under oxidative stress expressed high levels of the G1-specific CDK inhibitors p16Ink4a and p21Cip1 and senesced prematurely; this defect was corrected by knocking down CDK inhibitor levels with specific small interfering RNAs. These data suggest that YB-1 normally represses the transcription of CDK inhibitors, making it an important component of the cellular stress response signaling pathway. PMID:15899865

In vitro enzymatic reduction kinetics of mineral oxides by membrane fractions from Shewanella oneidensis MR-1

NASA Astrophysics Data System (ADS)

Ruebush, Shane S.; Icopini, Gary A.; Brantley, Susan L.; Tien, Ming

2006-01-01

This study documents the first example of in vitro solid-phase mineral oxide reduction by enzyme-containing membrane fractions. Previous in vitro studies have only reported the reduction of aqueous ions. Total membrane (TM) fractions from iron-grown cultures of Shewanella oneidensis MR-1 were isolated and shown to catalyze the reduction of goethite, hematite, birnessite, and ramsdellite/pyrolusite using formate. In contrast, nicotinamide adenine dinucleotide (NADH) and succinate cannot function as electron donors. The significant implications of observations related to this cell-free system are: (i) both iron and manganese mineral oxides are reduced by the TM fraction, but aqueous U(VI) is not; (ii) TM fractions from anaerobically grown, but not aerobically grown, cells can reduce the mineral oxides; (iii) electron shuttles and iron chelators are not needed for this in vitro reduction, documenting conclusively that reduction can occur by direct contact with the mineral oxide; (iv) electron shuttles and EDTA stimulate the in vitro Fe(III) reduction, documenting that exogenous molecules can enhance rates of enzymatic mineral reduction; and (v) multiple membrane components are involved in solid-phase oxide reduction. The membrane fractions, consisting of liposomes of cytoplasmic and outer membrane segments, contain at least 100 proteins including the enzyme that oxidizes formate, formate dehydrogenase. Mineral oxide reduction was inhibited by the addition of detergent Triton X-100, which solubilizes membranes and their associated proteins, consistent with the involvement of multiple electron carriers that are disrupted by detergent addition. In contrast, formate dehydrogenase activity was not inhibited by Triton X-100. The addition of anthraquinone-2,6-disulfonate (AQDS) and menaquinone-4 was unable to restore activity; however, menadione (MD) restored 33% of the activity. The addition of AQDS and MD to reactions without added detergent increased the rate of goethite reduction. The Michaelis-Menten Km values of 71 ± 22 m 2/L for hematite and 50 ± 16 m 2/L for goethite were calculated as a function of surface area of the two insoluble minerals. Vmax was determined to be 123 ± 14 and 156 ± 13 nmol Fe(II)/min/mg of TM protein for hematite and goethite, respectively. These values are consistent with in vivo rates of reduction reported in the literature. These observations are consistent with our conclusion that the enzymatic reduction of mineral oxides is an effective probe that will allow elucidation of molecular chemistry of the membrane-mineral interface where electron transfer occurs.

Kinetics of protein physicochemical changes induced by heating in meat using mimetic models: (1) relative effects of heat and oxidants.

PubMed

Promeyrat, A; Daudin, J D; Gatellier, P

2013-05-01

Optimizing the nutritional quality of cooked meat needs a better understanding of the mechanisms responsible for protein changes induced by heating. The relative contributions of chemical and thermal effects on protein physicochemical changes were studied using meat models. Two models were tested: a basic model made of an aqueous suspension of myofibrillar proteins, and a complex model, in which oxidants were added in physiological concentrations. Various heating time-temperature combinations were applied to both models in the ranges 45-90 °C and 5-120 min. Protein oxidation was evaluated by carbonyl and free thiol contents. Conformational changes of proteins were assessed by measurements of surface hydrophobicity and aggregation. Carbonyl formation was weakly affected by the thermal process alone but exacerbated by oxidants. A synergistic effect of oxidants and heat treatments on protein oxidation was noted. Changes in protein hydrophobicity and aggregation were dominated by the thermal process. Copyright © 2012 Elsevier Ltd. All rights reserved.

Iron(II) Initiation of Lipid and Protein Oxidation in Pork: The Role of Oxymyoglobin.

PubMed

Zhou, Feibai; Jongberg, Sisse; Zhao, Mouming; Sun, Weizheng; Skibsted, Leif H

2016-06-08

Iron(II), added as FeSO4·7H2O, was found to increase the rate of oxygen depletion as detected electrochemically in a pork homogenate from Longissimus dorsi through an initial increase in metmyoglobin formation from oxymyoglobin and followed by formation of primary and secondary lipid oxidation products and protein oxidation as detected as thiol depletion in myofibrillar proteins. Without added iron(II), under the same conditions at 37 °C, oxygen consumption corresponded solely to the slow oxymyoglobin autoxidation. Long-lived myofibrillar protein radicals as detected by ESR spectroscopy in the presence of iron(II) were formed subsequently to oxymyoglobin oxidation, and their level was increased by lipid oxidation when oxygen was completely depleted. Similarly, the time profile for formation of lipid peroxide indicated that oxymyoglobin oxidation initiates both protein oxidation and lipid oxidation.

High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis

PubMed Central

Aravena, Javier; Cabrera, Daniel; Simon, Felipe; Ezquer, Fernando

2016-01-01

Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs) are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes. PMID:27579157

O-GlcNAc and the Cardiovascular System

PubMed Central

Dassanayaka, Sujith; Jones, Steven P.

2014-01-01

The cardiovascular system is capable of robust changes in response to physiologic and pathologic stimuli through intricate signaling mechanisms. The area of metabolism has witnessed a veritable renaissance in the cardiovascular system. In particular, the post-translational β-O-linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents one such signaling pathway that has been implicated in the pathophysiology of cardiovascular disease. This highly dynamic protein modification may induce functional changes in proteins and regulate key cellular processes including translation, transcription, and cell death. In addition, its potential interplay with phosphorylation provides an additional layer of complexity to post-translational regulation. The hexosamine biosynthetic pathway generally requires glucose to form the nucleotide sugar, UDP-GlcNAc. Accordingly, O-GlcNAcylation may be altered in response to nutrient availability and cellular stress. Recent literature supports O-GlcNAcylation as an autoprotective response in models of acute stress (hypoxia, ischemia, oxidative stress). Models of sustained stress, such as pressure overload hypertrophy, and infarct-induced heart failure, may also require protein O-GlcNAcylation as a partial compensatory mechanism. Yet, in models of Type II diabetes, O-GlcNAcylation has been implicated in the subsequent development of vascular, and even cardiac, dysfunction. This review will address this apparent paradox and discuss the potential mechanisms of O-GlcNAc-mediated cardioprotection and cardiovascular dysfunction. This discussion will also address potential targets for pharmacologic interventions and the unique considerations related to such targets. PMID:24287310

O-GlcNAc and the cardiovascular system.

PubMed

Dassanayaka, Sujith; Jones, Steven P

2014-04-01

The cardiovascular system is capable of robust changes in response to physiologic and pathologic stimuli through intricate signaling mechanisms. The area of metabolism has witnessed a veritable renaissance in the cardiovascular system. In particular, the post-translational β-O-linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents one such signaling pathway that has been implicated in the pathophysiology of cardiovascular disease. This highly dynamic protein modification may induce functional changes in proteins and regulate key cellular processes including translation, transcription, and cell death. In addition, its potential interplay with phosphorylation provides an additional layer of complexity to post-translational regulation. The hexosamine biosynthetic pathway generally requires glucose to form the nucleotide sugar, UDP-GlcNAc. Accordingly, O-GlcNAcylation may be altered in response to nutrient availability and cellular stress. Recent literature supports O-GlcNAcylation as an autoprotective response in models of acute stress (hypoxia, ischemia, oxidative stress). Models of sustained stress, such as pressure overload hypertrophy, and infarct-induced heart failure, may also require protein O-GlcNAcylation as a partial compensatory mechanism. Yet, in models of Type II diabetes, O-GlcNAcylation has been implicated in the subsequent development of vascular, and even cardiac, dysfunction. This review will address this apparent paradox and discuss the potential mechanisms of O-GlcNAc-mediated cardioprotection and cardiovascular dysfunction. This discussion will also address potential targets for pharmacologic interventions and the unique considerations related to such targets. Copyright © 2013 Elsevier Inc. All rights reserved.

Modification and Functional Inhibition of Regulator of G-Protein Signaling 4 (RGS4) by 4-Hydroxy-2-nonenal

PubMed Central

Monroy, C. Aaron; Doorn, Jonathan A.; Roman, David L.

2015-01-01

Oxidative stress has been implicated as a component of various pathologies including ischemia/reperfusion injury (IRI) and neurodegenerative diseases such as Parkinson's disease (PD) and schizophrenia. Similarly, regulator of G-protein signaling 4 (RGS4) has been implicated as an important player in each of these pathologies. RGS4, like other RGS proteins, is responsible for temporally regulating G-protein coupled receptor signaling by increasing the intrinsic GTPase activity of Gα subunit of the heterotrimeric signaling complex. In this study we evaluated whether modification by 4-hydroxy-2-nonenal (4HNE), a common lipid peroxidation product, inhibits RGS4. Using immunoprecipitation, we first determined RGS4 modification was occurring in cells at concentrations of 4HNE within reported physiological conditions. Following this determination, we evaluated modification of RGS4 by 4HNE by both Western blot and mass spectrometry (MS). Once it was established that covalent modification occurred only on cysteine containing constructs, tryptic digest followed by mass spectrometry analysis revealed modification occurs at cysteine residues 71, 148, and 183. In order to determine the effect 4HNE had on RGS4 activity, a steady-state colorimetric assay was used to analyze the GAP activity of Δ51-RGS4 as well as the cysteine null mutant. From the data, we determined that RGS4 activity can be modulated by 4HNE through modification at cysteine residues similar to previously reported small molecule inhibition of RGS4. PMID:24229325

The retina/RPE proteome in chick myopia and hyperopia models: Commonalities with inherited and age-related ocular pathologies

PubMed Central

Riddell, Nina; Faou, Pierre; Murphy, Melanie; Giummarra, Loretta; Downs, Rachael A.; Rajapaksha, Harinda

2017-01-01

Purpose Microarray and RNA sequencing studies in the chick model of early optically induced refractive error have implicated thousands of genes, many of which have also been linked to ocular pathologies in humans, including age-related macular degeneration (AMD), choroidal neovascularization, glaucoma, and cataract. These findings highlight the potential relevance of the chick model to understanding both refractive error development and the progression to secondary pathological complications. The present study aimed to determine whether proteomic responses to early optical defocus in the chick share similarities with these transcriptome-level changes, particularly in terms of dysregulation of pathology-related molecular processes. Methods Chicks were assigned to a lens condition (monocular +10 D [diopters] to induce hyperopia, −10 D to induce myopia, or no lens) on post-hatch day 5. Biometric measures were collected following a further 6 h and 48 h of rearing. The retina/RPE was then removed and prepared for liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) on an LTQ-Orbitrap Elite. Raw data were processed using MaxQuant, and differentially abundant proteins were identified using moderated t tests (fold change ≥1.5, Benjamini-Hochberg adjusted p<0.05). These differentially abundant proteins were compared with the genes and proteins implicated in previous exploratory transcriptome and proteomic studies of refractive error, as well as the genes and proteins linked to the ocular pathologies listed above for which myopia or hyperopia are risk factors. Finally, gene set enrichment analysis (GSEA) was used to assess whether gene sets from the Human Phenotype Ontology database were enriched in the lens groups relative to the no lens groups, and at the top or bottom of the protein data ranked by Spearman’s correlation with refraction at 6 and 48 h. Results Refractive errors of −2.63 D ± 0.31 D (mean ± standard error, SE) and 3.90 D ± 0.37 D were evident in the negative and positive lens groups, respectively, at 6 h. By 48 h, refractive compensation to both lens types was almost complete (negative lens −9.70 D ± 0.41 D, positive lens 7.70 D ± 0.44 D). More than 140 differentially abundant proteins were identified in each lens group relative to the no lens controls at both time points. No proteins were differentially abundant between the negative and positive lens groups at 6 h, and 13 were differentially abundant at 48 h. As there was substantial overlap in the proteins implicated across the six comparisons, a total of 390 differentially abundant proteins were identified. Sixty-five of these 390 proteins had previously been implicated in transcriptome studies of refractive error animal models, and 42 had previously been associated with AMD, choroidal neovascularization, glaucoma, and/or cataract in humans. The overlap of differentially abundant proteins with AMD-associated genes and proteins was statistically significant for all conditions (Benjamini-Hochberg adjusted p<0.05), with over-representation analysis implicating ontologies related to oxidative stress, cholesterol homeostasis, and melanin biosynthesis. GSEA identified significant enrichment of genes associated with abnormal electroretinogram, photophobia, and nyctalopia phenotypes in the proteins negatively correlated with ocular refraction across the lens groups at 6 h. The implicated proteins were primarily linked to photoreceptor dystrophies and mitochondrial disorders in humans. Conclusions Optical defocus in the chicks induces rapid changes in the abundance of many proteins in the retina/RPE that have previously been linked to inherited and age-related ocular pathologies in humans. Similar changes have been identified in a meta-analysis of chick refractive error transcriptome studies, highlighting the chick as a model for the study of optically induced stress with possible relevance to understanding the development of a range of pathological states in humans. PMID:29259393

Detection and Identification of the Vibrational Markers for the Quantification of Methionine Oxidation in Therapeutic Proteins.

PubMed

Balakrishnan, Gurusamy; Barnett, Gregory V; Kar, Sambit R; Das, Tapan K

2018-05-17

Methionine oxidation is a major degradation pathway in therapeutic proteins which can impact the structure and function of proteins as well as risk to drug product quality. Detecting Met oxidation in proteins by peptide mapping followed by liquid chromatography with mass spectrometry (LC-MS) is the industry standard but is also labor intensive and susceptible to artifacts. In this work, vibrational difference spectroscopy in combination with 18 O isotopic shift enabled us to demonstrate the application of Raman and FTIR techniques for the detection and quantification of Met oxidation in various therapeutic proteins, including mAbs, fusion proteins, and antibody drug conjugate. Vibrational markers of Met oxidation products, such as sulfoxide and sulfone, corresponding to S═O and C-S═O stretching frequencies were unequivocally identified based 18 O isotoptic shifts. The intensity of the isolated νC-S Raman band at 702 cm -1 was successfully applied to quantify the average Met oxidation level in multiple proteins. These results are further corroborated by oxidation levels measured by tryptic peptide mapping, and thus the confirmed Met oxidation levels derived from Raman and mass spectrometry are indeed consistent with each other. Thus, we demonstrate the broader application of vibrational spectroscopy to detect the subtle spectral changes associated with various chemical or physical degradation of proteins, including Met oxidation as well as higher order structural changes.

Regulation of thrombosis and vascular function by protein methionine oxidation

PubMed Central

Gu, Sean X.; Stevens, Jeff W.

2015-01-01

Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis. PMID:25900980

Blood-Brain Barrier Disruption and Oxidative Stress in Guinea Pig after Systemic Exposure to Modified Cell-Free Hemoglobin

PubMed Central

Butt, Omer I.; Buehler, Paul W.; D'Agnillo, Felice

2011-01-01

Systemic exposure to cell-free hemoglobin (Hb) or its breakdown products after hemolysis or with the use of Hb-based oxygen therapeutics may alter the function and integrity of the blood-brain barrier. Using a guinea pig exchange transfusion model, we investigated the effect of a polymerized cell-free Hb (HbG) on the expression of endothelial tight junction proteins (zonula occludens 1, claudin-5, and occludin), astrocyte activation, IgG extravasation, heme oxygenase (HO), iron deposition, oxidative end products (4-hydroxynonenal adducts and 8-hydroxydeoxyguanosine), and apoptosis (cleaved caspase 3). Reduced zonula occludens 1 expression was observed after HbG transfusion as evidenced by Western blot and confocal microscopy. Claudin-5 distribution was altered in small- to medium-sized vessels. However, total expression of claudin-5 and occludin remained unchanged except for a notable increase in occludin 72 hours after HbG transfusion. HbG-transfused animals also showed increased astrocytic glial fibrillary acidic protein expression and IgG extravasation after 72 hours. Increased HO activity and HO-1 expression with prominent enhancement of HO-1 immunoreactivity in CD163-expressing perivascular cells and infiltrating monocytes/macrophages were also observed. Consistent with oxidative stress, HbG increased iron deposition, 4-hydroxynonenal and 8-hydroxydeoxyguanosine immunoreactivity, and cleaved caspase-3 expression. Systemic exposure to an extracellular Hb triggers blood-brain barrier disruption and oxidative stress, which may have important implications for the use of Hb-based therapeutics and may provide indirect insight on the central nervous system vasculopathies associated with excessive hemolysis. PMID:21356382

Selective Rac1 inhibition protects renal tubular epithelial cells from oxalate-induced NADPH oxidase-mediated oxidative cell injury

PubMed Central

Thamilselvan, Vijayalakshmi; Menon, Mani

2013-01-01

Oxalate-induced oxidative cell injury is one of the major mechanisms implicated in calcium oxalate nucleation, aggregation and growth of kidney stones. We previously demonstrated that oxalate-induced NADPH oxidase-derived free radicals play a significant role in renal injury. Since NADPH oxidase activation requires several regulatory proteins, the primary goal of this study was to characterize the role of Rac GTPase in oxalate-induced NADPH oxidase-mediated oxidative injury in renal epithelial cells. Our results show that oxalate significantly increased membrane translocation of Rac1 and NADPH oxidase activity of renal epithelial cells in a time-dependent manner. We found that NSC23766, a selective inhibitor of Rac1, blocked oxalate-induced membrane translocation of Rac1 and NADPH oxidase activity. In the absence of Rac1 inhibitor, oxalate exposure significantly increased hydrogen peroxide formation and LDH release in renal epithelial cells. In contrast, Rac1 inhibitor pretreatment, significantly decreased oxalate-induced hydrogen peroxide production and LDH release. Furthermore, PKC α and δ inhibitor, oxalate exposure did not increase Rac1 protein translocation, suggesting that PKC resides upstream from Rac1 in the pathway that regulates NADPH oxidase. In conclusion, our data demonstrate for the first time that Rac1-dependent activation of NADPH oxidase might be a crucial mechanism responsible for oxalate-induced oxidative renal cell injury. These findings suggest that Rac1 signaling plays a key role in oxalate-induced renal injury, and may serve as a potential therapeutic target to prevent calcium oxalate crystal deposition in stone formers and reduce recurrence. PMID:21814770

Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

PubMed

Sitte, N; Merker, K; von Zglinicki, T; Grune, T

2000-03-01

One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

Hydrogen sulfide (H2S) attenuates uranium-induced acute nephrotoxicity through oxidative stress and inflammatory response via Nrf2-NF-κB pathways.

PubMed

Zheng, Jifang; Zhao, Tingting; Yuan, Yan; Hu, Nan; Tang, Xiaoqing

2015-12-05

As an endogenous gaseous mediator, H2S exerts anti-oxidative, anti-inflammatory and cytoprotective effects in kidneys. This study was designed to investigate the protective effect of H2S against uranium-induced nephrotoxicity in adult SD male rats after in vivo effect of uranium on endogenous H2S formation was explored in kidneys. The levels of endogenous H2S and H2S-producing enzymes (CBS and CSE) were measured in renal homogenates from rats intoxicated by an intraperitoneally (i.p.) injection of uranyl acetate at a single dose of 2.5, 5 or 10 mg/kg. In rats injected i.p. with uranyl acetate (5 mg/kg) or NaHS (an H2S donor, 28 or 56 μmol/kg) alone or in combination, we determined biochemical parameters and histopathological alteration to assess kidney function, examined oxidative stress markers, and investigated Nrf2 and NF-κB pathways in kidney homogenates. The results suggest that uranium intoxication in rats decreased endogenous H2S generation as well as CBS and CSE protein expression. NaHS administration in uranium-intoxicated rats ameliorated the renal biochemical indices and histopathological effects, lowered MDA accumulation, and restored GSH level and anti-oxidative enzymes activities like SOD, CAT, GPx and GST. NaHS treatment in uranium-intoxicated rats activated uranium-inhibited protein expression and nuclear translocation of transcription factor Nrf2, which increased protein expression of downstream target-Nrf2 genes HO-1, NQO-1, GCLC, and TXNRD-1. NaHS administration in uranium-intoxicated rats inhibited uranium-induced nuclear translocation and phosphorylation of transcription factor κB/p65, which decreased protein expression of target-p65 inflammatory genes TNF-α, iNOS, and COX-2. Taken together, these data implicate that H2S can afford protection to rat kidneys against uranium-induced adverse effects through induction of antioxidant defense by activating Nrf2 pathway and reduction of inflammatory response by suppressing NF-κB pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Copper toxicity, oxidative stress, and antioxidant nutrients.

PubMed

Gaetke, Lisa M; Chow, Ching Kuang

2003-07-15

Copper (Cu) is an integral part of many important enzymes involved in a number of vital biological processes. Although normally bound to proteins, Cu may be released and become free to catalyze the formation of highly reactive hydroxyl radicals. Data obtained from in vitro and cell culture studies are largely supportive of Cu's capacity to initiate oxidative damage and interfere with important cellular events. Oxidative damage has been linked to chronic Cu-overload and/or exposure to excess Cu caused by accidents, occupational hazards, and environmental contamination. Additionally, Cu-induced oxidative damage has been implicated in disorders associated with abnormal Cu metabolism and neurodegenerative changes. Interestingly, a deficiency in dietary Cu also increases cellular susceptibility to oxidative damage. A number of nutrients have been shown to interact with Cu and alter its cellular effects. Vitamin E is generally protective against Cu-induced oxidative damage. While most in vitro or cell culture studies show that ascorbic acid aggravates Cu-induced oxidative damage, results obtained from available animal studies suggest that the compound is protective. High intakes of ascorbic acid and zinc may provide protection against Cu toxicity by preventing excess Cu uptake. Zinc also removes Cu from its binding site, where it may cause free radical formation. Beta-carotene, alpha-lipoic acid and polyphenols have also been shown to attenuate Cu-induced oxidative damage. Further studies are needed to better understand the cellular effects of this essential, but potentially toxic, trace mineral and its functional interaction with other nutrients.

Chronic exposure to high glucose impairs bradykinin-stimulated nitric oxide production by interfering with the phospholipase-C-implicated signalling pathway in endothelial cells: evidence for the involvement of protein kinase C.

PubMed

Tang, Y; Li, G D

2004-12-01

Overwhelming evidence indicates that endothelial cell dysfunction in diabetes is characterised by diminished endothelium-dependent relaxation, but the matter of the underlying molecular mechanism remains unclear. As nitric oxide (NO) production from the endothelium is the major player in endothelium-mediated vascular relaxation, we investigated the effects of high glucose on NO production, and the possible alterations of signalling pathways implicated in this scenario. NO production and intracellular Ca(2+) levels ([Ca(2+)](i)) were assessed using the fluorescent probes 4,5-diaminofluorescein diacetate and fura-2 respectively. Exposure of cultured bovine aortic endothelial cells to high glucose for 5 or 10 days significantly reduced NO production induced by bradykinin (but not by Ca(2+) ionophore) in a time- and dose-dependent manner. This was probably due to an attenuation in bradykinin-induced elevations of [Ca(2+)](i) under these conditions, since a close correlation between [Ca(2+)](i) increases and NO generation was observed in intact bovine aortic endothelial cells. Both bradykinin-promoted intracellular Ca(2+) mobilisation and extracellular Ca(2+) entry were affected. Moreover, bradykinin-induced formation of Ins(1,4,5)P(3), a phospholipase C product leading to increases in [Ca(2+)](i), was also inhibited following high glucose culture. This abnormality was not attributable to a decrease in inositol phospholipids, but possibly to a reduction in the number of bradykinin receptors. The alterations in NO production, the increases in [Ca(2+)](i), and the bradykinin receptor number due to high glucose could be largely reversed by protein kinase C inhibitors and D: -alpha-tocopherol (antioxidant). Chronic exposure to high glucose reduces NO generation in endothelial cells, probably by impairing phospholipase-C-mediated Ca(2+) signalling due to excess protein kinase C activation. This defect in NO release may contribute to the diminished endothelium-dependent relaxation and thus to the development of cardiovascular diseases in diabetes.

Toward an understanding of mechanism of aging-induced oxidative stress in human mesenchymal stem cells.

PubMed

Benameur, Laila; Charif, Naceur; Li, Yueying; Stoltz, Jean-François; de Isla, Natalia

2015-01-01

Under physiological conditions, there is a production of limited range of free radicals. However, when the cellular antioxidant defence systems, overwhelm and fail to reverse back the free radicals to their normal basal levels, there is a creation of a condition of redox disequilibrium termed "oxidative stress", which is implicated in a very wide spectrum of genetic, metabolic, and cellular responses. The excess of free radicals can, cause unfavourable molecular alterations to biomolecules through oxidation of lipids, proteins, RNA and DNA, that can in turn lead to mutagenesis, carcinogenesis, and aging. Mesenchymal stem cells (MSCs) have been proven to be a promising source of cells for regenerative medicine, and to be useful in the treatment of pathologies in which tissue damage is linked to oxidative stress. Moreover, MSCs appeared to efficiently manage oxidative stress and to be more resistant to oxidative insult than normal somatic cells, making them an interesting and testable model for the role of oxidative stress in the aging process. In addition, aging is accompanied by a progressive decline in stem cell function, resulting in less effective tissue homeostasis and repair. Also, there is an obvious link between intracellular reactive oxygen species levels and cellular senescence. To date, few studies have investigated the promotion of aging by oxidative stress on human MSCs, and the mechanism by which oxidative stress induce stem cell aging is poorly understood. In this context, the aim of this review is to gain insight the current knowledge about the molecular mechanisms of aging-induced oxidative stress in human MSCs.

Characteristic of the Oxidative Stress in Blood of Patients in Dependence of Community-Acquired Pneumonia Severity.

PubMed

Muravlyova, Larissa; Molotov-Luchankiy, Vilen; Bakirova, Ryszhan; Klyuyev, Dmitriy; Demidchik, Ludmila; Lee, Valentina

2016-03-15

At the present time the alternation of the oxidative metabolism is considered as one of the leading pathogenic mechanisms in the development and progression of community-acquired pneumonia (CAP). However the nature and direction of the oxidative protein changes in CAP patient's blood had been almost unexplored. To define oxidative and modified proteins in erythrocytes and blood plasma of CAP patients. Blood plasma and erythrocytes obtained from: 42 patients with moderate severity pneumonia, 12 patients with grave severity pneumonia and 32 healthy volunteers. Content of advanced oxidation protein products, malondialdehyde and reactive carbonyl derivatives were estimated as indicators of the oxidative stress and oxidative damage of proteins. In patients with grave severity the level of oxidative proteins and MDA in erythrocytes exceeded both: control values and similar meanings in CAP patients with moderate severity. The further growth of MDA in this group patients' blood plasma was observed, but the level of oxidative proteins decreased in comparison with those in CAP patients with moderate severity. To sum up, our derived data show, that injury of erythrocytes' redox-status and blood plasma components plays an essential role in development and progression CAP.

The role of β-adrenergic blockers in Parkinson's disease: possible genetic and cell-signaling mechanisms.

PubMed

Luong, Khanh vinh quoc; Nguyen, Lan Thi Hoàng

2013-06-01

Genetic studies have identified numerous factors linking β-adrenergic blockade to Parkinson's disease (PD), including human leukocyte antigen genes, the renin-angiotensin system, poly(adenosine diphosphate-ribose) polymerase 1, nerve growth factor, vascular endothelial growth factor, and the reduced form of nicotinamide adenine dinucleotide phosphate. β-Adrenergic blockade has also been implicated in PD via its effects on matrix metalloproteinases, mitogen-activated protein kinase pathways, prostaglandins, cyclooxygenase 2, and nitric oxide synthase. β-Adrenergic blockade may have a significant role in PD; therefore, the characterization of β-adrenergic blockade in patients with PD is needed.

Unique structure and regulation of the nematode detoxification gene regulator SKN-1: implications to understanding and controlling drug resistance

PubMed Central

Choe, Keith P.; Leung, Chi K.; Miyamoto, Michael M.

2012-01-01

Nematodes parasitize an alarming number of people and agricultural animals globally and cause debilitating morbidity and mortality. Anthelmintics have been the primary tools used to control parasitic nematodes for the past several decades, but drug resistance is becoming a major obstacle. Xenobiotic detoxification pathways defend against drugs and other foreign chemicals in diverse organisms, and evidence is accumulating that they play a role in mediating resistance to anthelmintics in nematodes. Related anti-oxidation pathways may also provide filarial parasites protection against host free radical-mediated immune responses. Upstream regulatory pathways have received almost no attention in nematode parasites despite their potential to co-regulate multiple detoxification and anti-oxidation genes. The NRF2 transcription factor mediates inducible detoxification and anti-oxidation defenses in mammals and recent studies have demonstrated that it promotes multidrug resistance in some human tumors. Recent studies in the free-living model nematode Caenorhabditis elegans have defined the homologous transcription factor SKN-1 as a master regulator of detoxification and anti-oxidation genes. Despite similar functions, SKN-1 and NRF2 have important differences in structure and regulatory pathways. Protein alignment and phylogenetic analyses indicate that these differences are shared among many nematodes making SKN-1 a candidate for specifically targeting nematode detoxification and anti-oxidation. PMID:22656429

Zinc and redox signaling: perturbations associated with cardiovascular disease and diabetes mellitus.

PubMed

Foster, Meika; Samman, Samir

2010-11-15

Cellular signal transduction pathways are influenced by the zinc and redox status of the cell. Numerous chronic diseases, including cardiovascular disease (CVD) and diabetes mellitus (DM), have been associated with impaired zinc utilization and increased oxidative stress. In humans, mutations in the MT-1A and ZnT8 genes, both of which are involved in the maintenance of zinc homeostasis, have been linked with DM development. Changes in levels of intracellular free zinc may exacerbate oxidative stress in CVD and DM by impacting glutathione homeostasis, nitric oxide signaling, and nuclear factor-kappa B-dependent cellular processes. Zinc ions have been shown to influence insulin and leptin signaling via the phosphoinositide 3′-kinase/Akt pathway, potentially linking an imbalance of zinc at the cellular level to insulin resistance and dyslipidemia. The oxidative modification of cysteine residues in zinc coordination sites in proteins has been implicated in cellular signaling and regulatory pathways. Despite the many interactions between zinc and cellular stress responses, studies investigating the potential therapeutic benefit of zinc supplementation in the prevention and treatment of oxidative stress-related chronic disease in humans are few and inconsistent. Further well-designed randomized controlled trials are needed to determine the effects of zinc supplementation in populations at various stages of CVD and DM progression.

The role of MAPK signal transduction pathways in the response to oxidative stress in the fungal pathogen Candida albicans: implications in virulence.

PubMed

de Dios, Carmen Herrero; Román, Elvira; Monge, Rebeca Alonso; Pla, Jesús

2010-12-01

In recent years, Mitogen-Activated Protein Kinase (MAPK) pathways have emerged as major regulators of cellular physiology. In the fungal pathogen Candida albicans, three different MAPK pathways have been characterized in the last years. The HOG pathway is mainly a stress response pathway that is activated in response to osmotic and oxidative stress and also participates regulating other pathways. The SVG pathway (or mediated by the Cek1 MAPK) is involved in cell wall formation under vegetative and filamentous growth, while the Mkc1-mediated pathway is involved in cell wall integrity. Oxidative stress is one of the types of stress that every fungal cell has to face during colonization of the host, where the cell encounters both hypoxia niches (i.e. gut) and high concentrations of reactive oxygen species (upon challenge with immune cells). Two pathways have been shown to be activated in response to oxidative stress: the HOG pathway and the MKC1-mediated pathway while the third, the Cek1 pathway is deactivated. The timing, kinetics, stimuli and functional responses generated upon oxidative stress differ among them; however, they have essential functional consequences that severely influence pathogenesis. MAPK pathways are, therefore, valuable targets to be explored in antifungal research.

Zidovudine Induces Downregulation of Mitochondrial Deoxynucleoside Kinases: Implications for Mitochondrial Toxicity of Antiviral Nucleoside Analogs

PubMed Central

Sun, Ren; Eriksson, Staffan

2014-01-01

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of deoxynucleosides in the synthesis of the DNA precursors required for mitochondrial DNA (mtDNA) replication and are essential for mitochondrial function. Antiviral nucleosides are known to cause toxic mitochondrial side effects. Here, we examined the effects of 3′-azido-2′,3′-dideoxythymidine (AZT) (zidovudine) on mitochondrial TK2 and dGK levels and found that AZT treatment led to downregulation of mitochondrial TK2 and dGK in U2OS cells, whereas cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) levels were not affected. The AZT effects on mitochondrial TK2 and dGK were similar to those of oxidants (e.g., hydrogen peroxide); therefore, we examined the oxidative effects of AZT. We found a modest increase in cellular reactive oxygen species (ROS) levels in the AZT-treated cells. The addition of uridine to AZT-treated cells reduced ROS levels and protein oxidation and prevented the degradation of mitochondrial TK2 and dGK. In organello studies indicated that the degradation of mitochondrial TK2 and dGK is a mitochondrial event. These results suggest that downregulation of mitochondrial TK2 and dGK may lead to decreased mitochondrial DNA precursor pools and eventually mtDNA depletion, which has significant implications for the regulation of mitochondrial nucleotide biosynthesis and for antiviral therapy using nucleoside analogs. PMID:25182642

Ascorbate protects endothelial barrier function during septic insult: Role of protein phosphatase type 2A.

PubMed

Han, Min; Pendem, Suresh; Teh, Suet Ling; Sukumaran, Dinesh K; Wu, Feng; Wilson, John X

2010-01-01

Endothelial barrier dysfunction contributes to morbidity in sepsis. We tested the hypothesis that raising the intracellular ascorbate concentration protects the endothelial barrier from septic insult by inhibiting protein phosphatase type 2A. Monolayer cultures of microvascular endothelial cells were incubated with ascorbate, dehydroascorbic acid (DHAA), the NADPH oxidase inhibitors apocynin and diphenyliodonium, or the PP2A inhibitor okadaic acid and then were exposed to septic insult (lipopolysaccharide and interferon-gamma). Under standard culture conditions that depleted intracellular ascorbate, septic insult stimulated oxidant production and PP2A activity, dephosphorylated phosphoserine and phosphothreonine residues in the tight junction-associated protein occludin, decreased the abundance of occludin at cell borders, and increased monolayer permeability to albumin. NADPH oxidase inhibitors prevented PP2A activation and monolayer leak, showing that these changes required reactive oxygen species. Okadaic acid, at a concentration that inhibited PP2A activity and monolayer leak, prevented occludin dephosphorylation and redistribution, implicating PP2A in the response of occludin to septic insult. Incubation with ascorbate or DHAA raised intracellular ascorbate concentrations and mitigated the effects of septic insult. In conclusion, ascorbate acts within microvascular endothelial cells to inhibit septic stimulation of oxidant production by NADPH oxidase and thereby prevents PP2A activation, PP2A-dependent dephosphorylation and redistribution of occludin, and disruption of the endothelial barrier. Copyright 2009 Elsevier Inc. All rights reserved.

Conservative and compensatory evolution in oxidative phosphorylation complexes of angiosperms with highly divergent rates of mitochondrial genome evolution.

PubMed

Havird, Justin C; Whitehill, Nicholas S; Snow, Christopher D; Sloan, Daniel B

2015-12-01

Interactions between nuclear and mitochondrial gene products are critical for eukaryotic cell function. Nuclear genes encoding mitochondrial-targeted proteins (N-mt genes) experience elevated rates of evolution, which has often been interpreted as evidence of nuclear compensation in response to elevated mitochondrial mutation rates. However, N-mt genes may be under relaxed functional constraints, which could also explain observed increases in their evolutionary rate. To disentangle these hypotheses, we examined patterns of sequence and structural evolution in nuclear- and mitochondrial-encoded oxidative phosphorylation proteins from species in the angiosperm genus Silene with vastly different mitochondrial mutation rates. We found correlated increases in N-mt gene evolution in species with fast-evolving mitochondrial DNA. Structural modeling revealed an overrepresentation of N-mt substitutions at positions that directly contact mutated residues in mitochondrial-encoded proteins, despite overall patterns of conservative structural evolution. These findings support the hypothesis that selection for compensatory changes in response to mitochondrial mutations contributes to the elevated rate of evolution in N-mt genes. We discuss these results in light of theories implicating mitochondrial mutation rates and mitonuclear coevolution as drivers of speciation and suggest comparative and experimental approaches that could take advantage of heterogeneity in rates of mtDNA evolution across eukaryotes to evaluate such theories. © 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.

Oxidative stress and inflammation in cerebral cavernous malformation disease pathogenesis: Two sides of the same coin.

PubMed

Retta, Saverio Francesco; Glading, Angela J

2016-12-01

Cerebral Cavernous Malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or is inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM lesions exhibit a range of different phenotypes, including wide inter-individual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Lesions may remain asymptomatic or result in pathological conditions of various type and severity at any age, with symptoms ranging from recurrent headaches to severe neurological deficits, seizures, and stroke. To date there are no direct therapeutic approaches for CCM disease besides the surgical removal of accessible lesions. Novel pharmacological strategies are particularly needed to limit disease progression and severity and prevent de novo formation of CCM lesions in susceptible individuals. Useful insights into innovative approaches for CCM disease prevention and treatment are emerging from a growing understanding of the biological functions of the three known CCM proteins, CCM1/KRIT1, CCM2 and CCM3/PDCD10. In particular, accumulating evidence indicates that these proteins play major roles in distinct signaling pathways, including those involved in cellular responses to oxidative stress, inflammation and angiogenesis, pointing to pathophysiological mechanisms whereby the function of CCM proteins may be relevant in preventing vascular dysfunctions triggered by these events. Indeed, emerging findings demonstrate that the pleiotropic roles of CCM proteins reflect their critical capacity to modulate the fine-tuned crosstalk between redox signaling and autophagy that govern cell homeostasis and stress responses, providing a novel mechanistic scenario that reconciles both the multiple signaling pathways linked to CCM proteins and the distinct therapeutic approaches proposed so far. In addition, recent studies in CCM patient cohorts suggest that genetic susceptibility factors related to differences in vascular sensitivity to oxidative stress and inflammation contribute to inter-individual differences in CCM disease susceptibility and severity. This review discusses recent progress into the understanding of the molecular basis and mechanisms of CCM disease pathogenesis, with specific emphasis on the potential contribution of altered cell responses to oxidative stress and inflammatory events occurring locally in the microvascular environment, and consequent implications for the development of novel, safe, and effective preventive and therapeutic strategies. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Gel-based methods in redox proteomics.

PubMed

Charles, Rebecca; Jayawardhana, Tamani; Eaton, Philip

2014-02-01

The key to understanding the full significance of oxidants in health and disease is the development of tools and methods that allow the study of proteins that sense and transduce changes in cellular redox. Oxidant-reactive deprotonated thiols commonly operate as redox sensors in proteins and a variety of methods have been developed that allow us to monitor their oxidative modification. This outline review specifically focuses on gel-based methods used to detect, quantify and identify protein thiol oxidative modifications. The techniques we discuss fall into one of two broad categories. Firstly, methods that allow oxidation of thiols in specific proteins or the global cellular pool to be monitored are discussed. These typically utilise thiol-labelling reagents that add a reporter moiety (e.g. affinity tag, fluorophore, chromophore), in which loss of labelling signifies oxidation. Secondly, we outline methods that allow specific thiol oxidation states of proteins (e.g. S-sulfenylation, S-nitrosylation, S-thionylation and interprotein disulfide bond formation) to be investigated. A variety of different gel-based methods for identifying thiol proteins that are sensitive to oxidative modifications have been developed. These methods can aid the detection and quantification of thiol redox state, as well as identifying the sensor protein. By understanding how cellular redox is sensed and transduced to a functional effect by protein thiol redox sensors, this will help us better appreciate the role of oxidants in health and disease. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Copyright © 2013 Elsevier B.V. All rights reserved.

The muscle fiber type–fiber size paradox: hypertrophy or oxidative metabolism?

PubMed Central

van Wessel, T.; de Haan, A.; van der Laarse, W. J.

2010-01-01

An inverse relationship exists between striated muscle fiber size and its oxidative capacity. This relationship implies that muscle fibers, which are triggered to simultaneously increase their mass/strength (hypertrophy) and fatigue resistance (oxidative capacity), increase these properties (strength or fatigue resistance) to a lesser extent compared to fibers increasing either of these alone. Muscle fiber size and oxidative capacity are determined by the balance between myofibrillar protein synthesis, mitochondrial biosynthesis and degradation. New experimental data and an inventory of critical stimuli and state of activation of the signaling pathways involved in regulating contractile and metabolic protein turnover reveal: (1) higher capacity for protein synthesis in high compared to low oxidative fibers; (2) competition between signaling pathways for synthesis of myofibrillar proteins and proteins associated with oxidative metabolism; i.e., increased mitochondrial biogenesis via AMP-activated protein kinase attenuates the rate of protein synthesis; (3) relatively higher expression levels of E3-ligases and proteasome-mediated protein degradation in high oxidative fibers. These observations could explain the fiber type–fiber size paradox that despite the high capacity for protein synthesis in high oxidative fibers, these fibers remain relatively small. However, it remains challenging to understand the mechanisms by which contractile activity, mechanical loading, cellular energy status and cellular oxygen tension affect regulation of fiber size. Therefore, one needs to know the relative contribution of the signaling pathways to protein turnover in high and low oxidative fibers. The outcome and ideas presented are relevant to optimizing treatment and training in the fields of sports, cardiology, oncology, pulmonology and rehabilitation medicine. Electronic supplementary material The online version of this article (doi:10.1007/s00421-010-1545-0) contains supplementary material, which is available to authorized users. PMID:20602111

Nutrition and muscle catabolism in maintenance hemodialysis: does feeding make muscle cells selective self-eaters?

PubMed

Franch, Harold A

2009-01-01

Efforts to build muscle by increased protein feeding in hemodialysis patients have been thwarted by parallel increases in both muscle protein synthesis and degradation. The evidence suggests that muscle cells replace older proteins in response to feeding rather than using new proteins to drive muscle cell hypertrophy. This review presents the hypothesis that protein feeding provides an opportunity for muscle to accelerate proteolysis of proteins that have been damaged by oxidation, nitrosylation, and/or glycosylation and to replace damaged mitochondria that contribute to oxidative stress. Increases in proteolysis with feeding are driven by insulin resistance and the increased oxidative stress of mitochondrial respiration. Oxidized proteins and organelles are excellent substrates for degradation by the proteasome, macroautophagy, and chaperone-mediated autophagy: these systems of proteolysis seem to be activated by oxydatiative stress. Replacement of oxidized and other damaged proteins may be a benefit of protein feeding in hemodialysis, but alternative strategies, including exercise, will be required to build muscle.

Nutrition and Muscle Catabolism in Maintenance Hemodialysis: Does Feeding Make Muscle Cells Selective Self-Eaters?

PubMed Central

Franch, Harold A.

2009-01-01

Efforts to build muscle by increased protein feeding in hemodialysis patients have been thwarted by parallel increases in both muscle protein synthesis and degradation. The evidence suggests that muscle cells replace older proteins in response to feeding rather than using new proteins to drive muscle cell hypertrophy. This review presents the hypothesis that protein feeding provides an opportunity for muscle to accelerate proteolysis of proteins which have been damaged by oxidation, nitrosylation and/or glycosylation and to replace damaged mitochondria that contribute to oxidative stress. Increases in proteolysis with feeding are driven by insulin resistance and the increased oxidative stress of mitochondrial respiration. Oxidized proteins and organelles are excellent substrates for degradation by the proteasome, macroautophagy, and chaperone-mediated autophagy: these systems of proteolysis seem to be activated by oxydatiative stress. Replacement of oxidized and other damaged proteins may be a benefit of protein feeding in hemodialysis, but alternative strategies, including exercise, will be required to build muscle. PMID:19121779

Redox Reactivity of Cerium Oxide Nanoparticles Induces the Formation of Disulfide Bridges in Thiol-Containing Biomolecules.

PubMed

Rollin-Genetet, Françoise; Seidel, Caroline; Artells, Ester; Auffan, Mélanie; Thiéry, Alain; Vidaud, Claude

2015-12-21

The redox state of disulfide bonds is implicated in many redox control systems, such as the cysteine-cystine couple. Among proteins, ubiquitous cysteine-rich metallothioneins possess thiolate metal binding groups susceptible to metal exchange in detoxification processes. CeO2 NPs are commonly used in various industrial applications due to their redox properties. These redox properties that enable dual oxidation states (Ce(IV)/Ce(III)) to exist at their surface may act as oxidants for biomolecules. The interaction among metallothioneins, cysteine, and CeO2 NPs was investigated through various biophysical approaches to shed light on the potential effects of the Ce(4+)/Ce(3+) redox system on the thiol groups of these biomolecules. The possible reaction mechanisms include the formation of a disulfide bridge/Ce(III) complex resulting from the interaction between Ce(IV) and the thiol groups, leading to metal unloading from the MTs, depending on their metal content and cluster type. The formation of stable Ce(3+) disulfide complexes has been demonstrated via their fluorescence properties. This work provides the first evidence of thiol concentration-dependent catalytic oxidation mechanisms between pristine CeO2 NPs and thiol-containing biomolecules.

An unexpected role for the yeast nucleotide exchange factor Sil1 as a reductant acting on the molecular chaperone BiP

PubMed Central

Siegenthaler, Kevin D; Pareja, Kristeen A; Wang, Jie; Sevier, Carolyn S

2017-01-01

Unfavorable redox conditions in the endoplasmic reticulum (ER) can decrease the capacity for protein secretion, altering vital cell functions. While systems to manage reductive stress are well-established, how cells cope with an overly oxidizing ER remains largely undefined. In previous work (Wang et al., 2014), we demonstrated that the chaperone BiP is a sensor of overly oxidizing ER conditions. We showed that modification of a conserved BiP cysteine during stress beneficially alters BiP chaperone activity to cope with suboptimal folding conditions. How this cysteine is reduced to reestablish 'normal' BiP activity post-oxidative stress has remained unknown. Here we demonstrate that BiP's nucleotide exchange factor – Sil1 – can reverse BiP cysteine oxidation. This previously unexpected reductant capacity for yeast Sil1 has potential implications for the human ataxia Marinesco-Sjögren syndrome, where it is interesting to speculate that a disruption in ER redox-signaling (due to genetic defects in SIL1) may influence disease pathology. DOI: http://dx.doi.org/10.7554/eLife.24141.001 PMID:28257000

Identification, expression and function of apolipoprotein E in annual fish Nothobranchius guentheri: implication for an aging marker.

PubMed

Wang, Xia; Shang, Xiaomei; Luan, Jing; Zhang, Shicui

2014-06-01

Apolipoprotein E (apoE) is a lipid-associated protein present in both plasma and in central nervous system. Variation in apoE gene has been reported to be associated with longevity in humans as well as with aged diseases such as atherosclerosis, Alzheimer's disease, and Parkinson's disease. However, information regarding the function and structure-activity relationship of apoE in lower vertebrates is rather limited. In this study we show that the apoE gene from the annual fish Nothobranchius guentheri, NapoE, encodes a protein of 262 amino acids, which shares common structural features characteristic of mammalian apoE. We also show that like human apoE, recombinant NapoE is able to inhibit LDL oxidation, and it is the N-terminal domain of NapoE with lysine or arginine residues that plays a key role in inhibition of LDL oxidation. NapoE is predominantly expressed in the liver of N. guentheri, consistent with that in mammalian species. More importantly, we demonstrate an age-dependent down-regulation of NapoE gene, rendering it a suitable biomarker of aging. This lays a foundation for further study of the role of apoE in the aging process of fish.

Human frataxin is an allosteric switch that activates the Fe-S cluster biosynthetic complex.

PubMed

Tsai, Chi-Lin; Barondeau, David P

2010-11-02

Cellular depletion of the human protein frataxin is correlated with the neurodegenerative disease Friedreich's ataxia and results in the inactivation of Fe-S cluster proteins. Most researchers agree that frataxin functions in the biogenesis of Fe-S clusters, but its precise role in this process is unclear. Here we provide in vitro evidence that human frataxin binds to a Nfs1, Isd11, and Isu2 complex to generate the four-component core machinery for Fe-S cluster biosynthesis. Frataxin binding dramatically changes the K(M) for cysteine from 0.59 to 0.011 mM and the catalytic efficiency (k(cat)/K(M)) of the cysteine desulfurase from 25 to 7900 M⁻¹s⁻¹. Oxidizing conditions diminish the levels of both complex formation and frataxin-based activation, whereas ferrous iron further stimulates cysteine desulfurase activity. Together, these results indicate human frataxin functions with Fe(2+) as an allosteric activator that triggers sulfur delivery and Fe-S cluster assembly. We propose a model in which cellular frataxin levels regulate human Fe-S cluster biosynthesis that has implications for mitochondrial dysfunction, oxidative stress response, and both neurodegenerative and cardiovascular disease.

Epigallocatechin activates haem oxygenase-1 expression via protein kinase Cδ and Nrf2

PubMed Central

Ogborne, Richard M.; Rushworth, Stuart A.; O’Connell, Maria A.

2008-01-01

The Nrf2/anti-oxidant response element (ARE) pathway plays an important role in regulating cellular anti-oxidants, including haem oxygenase-1 (HO-1). Various kinases have been implicated in the pathways leading to Nrf2 activation. Here, we investigated the effect of epigallocatechin (EGC) on ARE-mediated gene expression in human monocytic cells. EGC time and dose dependently increased HO-1 mRNA and protein expression but had minimal effect on expression of other ARE-regulated genes, including NAD(P)H:quinone oxidoreductase 1, glutathione cysteine ligase and ferritin. siRNA knock down of Nrf2 significantly inhibited EGC-induced HO-1 expression. Furthermore, inhibition of PKC by Ro-31-8220 dose dependently decreased EGC-induced HO-1 mRNA expression, whereas MAP kinase and phosphatidylinositol-3-kinase pathway inhibitors had no significant effect. EGC stimulated phosphorylation of PKCαβ and δ in THP-1 cells. PKCδ inhibition significantly decreased EGC-induced HO-1 mRNA expression, whereas PKCα- and β-specific inhibitors had no significant effect. These results demonstrate for the first time that EGC-induced HO-1 expression occurs via PKCδ and Nrf2. PMID:18586007

Insight into the molecular mechanism of the sulfur oxidation process by reverse sulfite reductase (rSiR) from sulfur oxidizer Allochromatium vinosum.

PubMed

Ghosh, Semanti; Bagchi, Angshuman

2018-04-26

Sulfur metabolism is one of the oldest known biochemical processes. Chemotrophic or phototrophic proteobacteria, through the dissimilatory pathway, use sulfate, sulfide, sulfite, thiosulfate or elementary sulfur by either reductive or oxidative mechanisms. During anoxygenic photosynthesis, anaerobic sulfur oxidizer Allochromatium vinosum forms sulfur globules that are further oxidized by dsr operon. One of the key redox enzymes in reductive or oxidative sulfur metabolic pathways is the DsrAB protein complex. However, there are practically no reports to elucidate the molecular mechanism of the sulfur oxidation process by the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum. In the present context, we tried to analyze the structural details of the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum by molecular dynamics simulations. The molecular dynamics simulation results revealed the various types of molecular interactions between DsrA and DsrB proteins during the formation of DsrAB protein complex. We, for the first time, predicted the mode of binding interactions between the co-factor and DsrAB protein complex from Allochromatium vinosum. We also compared the binding interfaces of DsrAB from sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris. This study is the first to provide a comparative aspect of binding modes of sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris.

Protective Effects of Combined Selenium and Punica granatum Treatment on Some Inflammatory and Oxidative Stress Markers in Arsenic-Induced Hepatotoxicity in Rats.

PubMed

Shafik, Noha M; El Batsh, Maha M

2016-01-01

Oxidative stress is one of the major mechanisms implicated in inorganic arsenic poisoning. Punica granatum is known by its free radical scavenging properties. The aim of this study was to evaluate the protective role of combined selenium and P. granatum against arsenic-induced liver injury. Seventy-five female albino rats were divided into five groups (of 15 rats each). Toxicity was induced by oral sodium arsenite (5.5 mg/kg body weight (bw) daily) (group ІІ). Treatment of arsenic-intoxicated rats was induced by daily oral administration of sodium selenite (3 mg/kg bw) (group ІІІ), 100 mg of P. granatum ethanol extract per kilogram body weight dissolved in 300 mL distilled water in three divided doses (100 mL of this suspension every 8 h) (group IV), and combined daily oral treatment with both selenite and P. granatum ethanol extract (group V). After 3 weeks, serum and liver tissues were obtained from the decapitated rats for different estimations. Hepatotoxicity was demonstrated by significant elevation in liver weights and activities of liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and decrease in serum total proteins and albumin (p < 0.05) which were confirmed by histopathological examination. Additionally, arsenic hepatotoxicity led to an increased values of malondialdehyde, advanced oxidation protein products, nitric oxide, and interleukin-6 (IL-6) (p < 0.05) and decreased activity of thioredoxin reductase, values of total anti-oxidant capacity, and nuclear factor erythroid 2-related factor 2 (Nrf2) gene expression. Significant improvement in all assessed parameters was observed in rat group treated with both P. granatum and selenium. It was concluded that combined P. granatum and selenium treatment had a synergistic hepatoprotective effect against arsenic toxicity through activation of Nrf2 anti-oxidant pathway.

The production of nitric oxide in EL4 lymphoma cells overexpressing growth hormone.

PubMed

Arnold, Robyn E; Weigent, Douglas A

2003-01-01

Growth hormone (GH) is produced by immunocompetent cells and has been implicated in the regulation of a multiplicity of functions in the immune system involved in growth and activation. However, the actions of endogenous or lymphocyte GH and its contribution to immune reactivity when compared with those of serum or exogenous GH are still unclear. In the present study, we overexpressed lymphocyte GH in EL4 lymphoma cells, which lack the GH receptor (GHR), to determine the role of endogenous GH in nitric oxide (NO) production and response to genotoxic stress. Western blot analysis demonstrated that the levels of GH increased approximately 40% in cells overexpressing GH (GHo) when compared with cells with vector alone. The results also show a substantial increase in NO production in cells overexpressing GH that could be blocked by N(G)-monomethyl-L-arginine (L-NMMA), an L-arginine analogue that competitively inhibits all three isoforms of nitric oxide synthase (NOS). No evidence was obtained to support an increase in peroxynitrite in cells overexpressing GH. Overexpression of GH increased NOS activity, inducible nitric oxide synthase (iNOS) promoter activity, and iNOS protein expression, whereas endothelial nitric oxide synthase and neuronal nitric oxide synthase protein levels were essentially unchanged. In addition, cells overexpressing GH showed increased arginine transport ability and intracellular arginase activity when compared with control cells. GH overexpression appeared to protect cells from the toxic effects of the DNA alkylating agent methyl methanesulfonate. This possibility was suggested by maintenance of the mitochondrial transmembrane potential in cells overexpressing GH when compared with control cells that could be blocked by L-NMMA. Taken together, the data support the notion that lymphocyte GH, independently of the GH receptor, may play a key role in the survival of lymphocytes exposed to stressful stimuli via the production of NO.

Nitric Oxide and Protein S-Nitrosylation Are Integral to Hydrogen Peroxide-Induced Leaf Cell Death in Rice1[W][OA

PubMed Central

Lin, Aihong; Wang, Yiqin; Tang, Jiuyou; Xue, Peng; Li, Chunlai; Liu, Linchuan; Hu, Bin; Yang, Fuquan; Loake, Gary J.; Chu, Chengcai

2012-01-01

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H2O2) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H2O2-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H2O2-induced leaf cell death in rice. PMID:22106097

Acylcarnitine Profiles in Acetaminophen Toxicity in the Mouse: Comparison to Toxicity, Metabolism and Hepatocyte Regeneration

PubMed Central

Bhattacharyya, Sudeepa; Pence, Lisa; Beger, Richard; Chaudhuri, Shubhra; McCullough, Sandra; Yan, Ke; Simpson, Pippa; Hennings, Leah; Hinson, Jack; James, Laura

2013-01-01

High doses of acetaminophen (APAP) result in hepatotoxicity that involves metabolic activation of the parent compound, covalent binding of the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) to liver proteins, and depletion of hepatic glutathione. Impaired fatty acid β-oxidation has been implicated in previous studies of APAP-induced hepatotoxicity. To better understand relationships between toxicity and fatty acid β-oxidation in the liver in APAP toxicity, metabolomic assays for long chain acylcarnitines were examined in relationship to established markers of liver toxicity, oxidative metabolism, and liver regeneration in a time course study in mice. Male B6C3F1 mice were treated with APAP (200 mg/kg IP) or saline and sacrificed at 1, 2, 4, 8, 24 or 48 h after APAP. At 1 h, hepatic glutathione was depleted and APAP protein adducts were markedly increased. Alanine aminotransferase (ALT) levels were elevated at 4 and 8 h, while proliferating cell nuclear antigen (PCNA) expression, indicative of hepatocyte regeneration, was apparent at 24 h and 48 h. Elevations of palmitoyl, oleoyl and myristoyl carnitine were apparent by 2–4 h, concurrent with the onset of Oil Red O staining in liver sections. By 8 h, acylcarnitine levels were below baseline levels and remained low at 24 and 48 h. A partial least squares (PLS) model suggested a direct association of acylcarnitine accumulation in serum to APAP protein adduct and hepatic glutathione levels in mice. Overall, the kinetics of serum acylcarnitines in APAP toxicity in mice followed a biphasic pattern involving early elevation after the metabolism phases of toxicity and later depletion of acylcarnitines. PMID:24958141

The role of SIRT1 in diabetic retinopathy.

PubMed

Karbasforooshan, Hedyieh; Karimi, Gholamreza

2018-01-01

The prevalence of diabetes mellitus (DM), has been increasing worldwide. Diabetic retinopathy (DR) is the most common microvascular complication in diabetes. It is a multifactorial disease that occurs primarily through the long-term detrimental effects of hyperglycemia. The pathogenesis of DR is complex, including inflammation, oxidative stress and advanced glycation end products (AGES). SIRT1 is a nicotinamide adenosine dinucleotide (NAD+)-dependent deacetylase that removes acetyl groups from proteins which can be implicated in DR. Inhibition of miRNAs such as miR-23b- 3P and miR-34a and activation of adenosine monophosphate-activated protein kinas (AMPK) and Peroxisome proliferative-activated receptor α (PPARα), modulate inflammation by enhancing the level of SIRT1. SIRT1 activation leads to the down regulation of nuclear factor kappa-light-chain-enhancer of activated of B cells (NF-κB), and the downstream pathway including increased level of Interleukin-17 (IL-17) and other pro-inflammatory cytokines. Oxidative stress- induced apoptosis is due to activation of some transcriptional factors such as p53 and Protein arginine methyl transferase 1 (PRMIT1) which are inhibited by SIRT1. In addition to these, the increased level of some transcriptional factors such as, vascular endothelial growth factor (VEGF), hypoxia - induced factors (HIFs), transforming growth factor β1 (TGF-β1), endothelin-1 (ET-1), fork head box O 1 (FOXO1) and Notch signaling may be inhibited by activation of SIRT1 leads to attenuation of vascular dysfunction. In conclusion, SIRT1 regulates apoptosis, inflammation and oxidative stress resulting in improving DR. This review focuses on the role of SIRT1 in DR. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The effect of β-N-methylamino-L-alanine (BMAA) on oxidative stress response enzymes of the macrophyte Ceratophyllum demersum.

PubMed

Esterhuizen-Londt, M; Pflugmacher, S; Downing, T G

2011-04-01

Cyanobacteria are known to produce bioactive secondary metabolites such as hepatotoxins, cytotoxins and neurotoxins. The newly recognized neurotoxin β-N-methylamino-L-alanine (BMAA) is a naturally occurring non-protein amino acid found in the majority of cyanobacterial genera tested. Evidence that exists for implication of BMAA in neurodegenerative disorders relies on bioaccumulation and biomagnification from symbiotic cyanobacteria. Uptake and accumulation of free BMAA by various non-symbiotic organisms, including aquatic macrophytes, has been documented but to date limited evidence of ecotoxicology exists. We therefore investigated the effect of BMAA on the oxidative stress responses of the macrophyte, Ceratophyllum demersum. Markers for oxidative stress in this study are the antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase and glutathione reductase. We found that BMAA had an inhibitory effect on all the oxidative stress response enzymes tested in plants exposed to BMAA. However enzymes not related to oxidative stress response were not affected by BMAA in in vitro experiments. Binding studies in the presence of BMAA showed reduced enzyme specific activity over time compared to the control. This study shows that BMAA causes oxidative stress indirectly as it inhibits antioxidant enzymes required to combat reactive oxygen species that cause damage to cells. Further investigations are required to fully understand the inhibitory effect of BMAA on these enzymes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Increase in peripheral oxidative stress during hypercholesterolemia is not reflected in the central nervous system: evidence from two mouse models.

PubMed

Ding, Tao; Yao, Yeumang; Praticò, Domenico

2005-05-01

In recent years oxidative stress has been widely implicated as a pathogenetic mechanism of several diseases, and a variety of indices and assays have been developed to assess this phenomenon in complex biological systems. Most of these biomarkers can be measured virtually in every biological fluid and tissue, providing us with the opportunity to assess their formation at local site of oxidative injury. However, despite their widespread use, it is still not completely clear how their peripheral formation correlates with the levels measured in the central nervous system. For this reason, we utilized two well-characterized animal models of chronic peripheral oxidative stress, low-density lipoprotein receptor (LDLR)-deficient and C57BL/6 mice on a high fat diet. After 8 weeks on the diet, we assessed isoprostane, marker of lipid peroxidation, and carbonyls, marker of protein oxidation, in several organs of these animals. Compared with animals on chow, mice on the high fat diet showed a significant increase in both biomarkers in plasma, heart, aorta and liver but not in brain tissues. This observation was confirmed by the selective accumulation of radioactivity in the peripheral organs but not in the brains of mice injected with tritiated isoprostane. Our findings indicate that in hypercholesterolemia the peripheral formation of oxidative products does not contribute to their levels found in the central nervous system.

Reproduction Is Associated with a Tissue-Dependent Reduction of Oxidative Stress in Eusocial Female Damaraland Mole-Rats (Fukomys damarensis)

PubMed Central

Schmidt, Christina M.; Blount, Jonathan D.; Bennett, Nigel C.

2014-01-01

Oxidative stress has been implicated as both a physiological cost of reproduction and a driving force on an animal's lifespan. Since increased reproductive effort is generally linked with a reduction in survival, it has been proposed that oxidative stress may influence this relationship. Support for this hypothesis is inconsistent, but this may, in part, be due to the type of tissues that have been analyzed. In Damaraland mole-rats the sole reproducing female in the colony is also the longest lived. Therefore, if oxidative stress does impact the trade-off between reproduction and survival in general, this species may possess some form of enhanced defense. We assessed this relationship by comparing markers of oxidative damage (malondialdehyde, MDA; protein carbonyls, PC) and antioxidants (total antioxidant capacity, TAC; superoxide dismutase, SOD) in various tissues including plasma, erythrocytes, heart, liver, kidney and skeletal muscle between wild-caught reproductive and non-reproductive female Damaraland mole-rats. Reproductive females exhibited significantly lower levels of PC across all tissues, and lower levels of MDA in heart, kidney and liver relative to non-reproductive females. Levels of TAC and SOD did not differ significantly according to reproductive state. The reduction in oxidative damage in breeding females may be attributable to the unusual social structure of this species, as similar relationships have been observed between reproductive and non-reproductive eusocial insects. PMID:25068591

A brain proteomic investigation of rapamycin effects in the Tsc1+/- mouse model.

PubMed

Wesseling, Hendrik; Elgersma, Ype; Bahn, Sabine

2017-01-01

Tuberous sclerosis complex (TSC) is a rare monogenic disorder characterized by benign tumors in multiple organs as well as a high prevalence of epilepsy, intellectual disability and autism. TSC is caused by inactivating mutations in the TSC1 or TSC2 genes. Heterozygocity induces hyperactivation of mTOR which can be inhibited by mTOR inhibitors, such as rapamycin, which have proven efficacy in the treatment of TSC-associated symptoms. The aim of the present study was (1) to identify molecular changes associated with social and cognitive deficits in the brain tissue of Tsc1 +/- mice and (2) to investigate the molecular effects of rapamycin treatment, which has been shown to ameliorate genotype-related behavioural deficits. Molecular alterations in the frontal cortex and hippocampus of Tsc1 +/- and control mice, with or without rapamycin treatment, were investigated. A quantitative mass spectrometry-based shotgun proteomic approach (LC-MS E ) was employed as an unbiased method to detect changes in protein levels. Changes identified in the initial profiling stage were validated using selected reaction monitoring (SRM). Protein Set Enrichment Analysis was employed to identify dysregulated pathways. LC-MS E analysis of Tsc1 +/- mice and controls ( n  = 30) identified 51 proteins changed in frontal cortex and 108 in the hippocampus. Bioinformatic analysis combined with targeted proteomic validation revealed several dysregulated molecular pathways. Using targeted assays, proteomic alterations in the hippocampus validated the pathways "myelination", "dendrite," and "oxidative stress", an upregulation of ribosomal proteins and the mTOR kinase. LC-MS E analysis was also employed on Tsc1 +/- and wildtype mice ( n  = 34) treated with rapamycin or vehicle. Rapamycin treatment exerted a stronger proteomic effect in Tsc1 +/- mice with significant changes (mainly decreased expression) in 231 and 106 proteins, respectively. The cellular pathways "oxidative stress" and "apoptosis" were found to be affected in Tsc1 +/- mice and the cellular compartments "myelin sheet" and "neurofilaments" were affected by rapamycin treatment. Thirty-three proteins which were altered in Tsc1 +/- mice were normalized following rapamycin treatment, amongst them oxidative stress related proteins, myelin-specific and ribosomal proteins. Molecular changes in the Tsc1 +/- mouse brain were more prominent in the hippocampus compared to the frontal cortex. Pathways linked to myelination and oxidative stress response were prominently affected and, at least in part, normalized following rapamycin treatment. The results could aid in the identification of novel drug targets for the treatment of cognitive, social and psychiatric symptoms in autism spectrum disorders. Similar pathways have also been implicated in other psychiatric and neurodegenerative disorders and could imply similar disease processes. Thus, the potential efficacy of mTOR inhibitors warrants further investigation not only for autism spectrum disorders but also for other neuropsychiatric and neurodegenerative diseases.

Sulphur Atoms from Methionines Interacting with Aromatic Residues Are Less Prone to Oxidation

PubMed Central

Aledo, Juan C.; Cantón, Francisco R.; Veredas, Francisco J.

2015-01-01

Methionine residues exhibit different degrees of susceptibility to oxidation. Although solvent accessibility is a relevant factor, oxidation at particular sites cannot be unequivocally explained by accessibility alone. To explore other possible structural determinants, we assembled different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins. Comparisons of the proteins containing oxidized methionines with all proteins in the human proteome led to the conclusion that the former exhibit a significantly higher mean value of methionine content than the latter. Within a given protein, an examination of the sequence surrounding the non-oxidized methionine revealed a preference for neighbouring tyrosine and tryptophan residues, but not for phenylalanine residues. However, because the interaction between sulphur atoms and aromatic residues has been reported to be important for the stabilization of protein structure, we carried out an analysis of the spatial interatomic distances between methionines and aromatic residues, including phenylalanine. The results of these analyses uncovered a new determinant for methionine oxidation: the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants. PMID:26597773

Occurrence State and Molecular Structure Analysis of Extracellular Proteins with Implications on the Dewaterability of Waste-Activated Sludge.

PubMed

Wu, Boran; Ni, Bing-Jie; Horvat, Kristine; Song, Liyan; Chai, Xiaoli; Dai, Xiaohu; Mahajan, Devinder

2017-08-15

The occurrence state and molecular structure of extracellular proteins were analyzed to reveal the influencing factors on the water-holding capacities of protein-like substances in waste-activated sludge (WAS). The gelation process of extracellular proteins verified that advanced oxidation processes (AOPs) for WAS dewaterability improvement eliminated the water affinity of extracellular proteins and prevented these macromolecules from forming stable colloidal aggregates. Isobaric tags for relative and absolute quantitation proteomics identified that most of the extracellular proteins were originally derived from the intracellular part and the proteins originally located in the extracellular part were mainly membrane-associated. The main mechanism of extracellular protein transformation during AOPs could be represented by the damage of the membrane or related external encapsulating structure and the release of intracellular substances. For the selected representative extracellular proteins, the strong correlation (R 2 > 0.97, p < 0.03) between the surface hydrophilicity index and α-helix percentages in the secondary structure indicated that the water affinity relied more on the spatial distribution of hydrophilic functional groups rather than the content. Destructing the secondary structure represented by the α-helix and stretching the polypeptide aggregation in the water phase through disulfide bond removal might be the key to eliminating the inhibitory effects of extracellular proteins on the interstitial water removal from WAS.

Selectivity of protein oxidative damage during aging in Drosophila melanogaster.

PubMed Central

Das, N; Levine, R L; Orr, W C; Sohal, R S

2001-01-01

The purpose of the present study was to determine whether oxidation of various proteins during the aging process occurs selectively or randomly, and whether the same proteins are damaged in different species. Protein oxidative damage to the proteins, present in the matrix of mitochondria in the flight muscles of Drosophila melanogaster and manifested as carbonyl modifications, was detected immunochemically with anti-dinitrophenyl-group antibodies. Aconitase was found to be the only protein in the mitochondrial matrix that exhibited an age-associated increase in carbonylation. The accrual of oxidative damage was accompanied by an approx. 50% loss in aconitase activity. An increase in ambient temperature, which elevates the rate of metabolism and shortens the life span of flies, caused an elevation in the amount of aconitase carbonylation and an accelerated loss in its activity. Exposure to 100% ambient oxygen showed that aconitase was highly susceptible to undergo oxidative damage and loss of activity under oxidative stress. Administration of fluoroacetate, a competitive inhibitor of aconitase activity, resulted in a dose-dependent decrease in the life span of the flies. Results of the present study demonstrate that protein oxidative damage during aging is a selective phenomenon, and might constitute a mechanism by which oxidative stress causes age-associated losses in specific biochemical functions. PMID:11696009

Regulation of thrombosis and vascular function by protein methionine oxidation.

PubMed

Gu, Sean X; Stevens, Jeff W; Lentz, Steven R

2015-06-18

Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis. © 2015 by The American Society of Hematology.

Previous physical exercise alters the hepatic profile of oxidative-inflammatory status and limits the secondary brain damage induced by severe traumatic brain injury in rats.

PubMed

de Castro, Mauro Robson Torres; Ferreira, Ana Paula de Oliveira; Busanello, Guilherme Lago; da Silva, Luís Roberto Hart; da Silveira Junior, Mauro Eduardo Porto; Fiorin, Fernando da Silva; Arrifano, Gabriela; Crespo-López, Maria Elena; Barcelos, Rômulo Pillon; Cuevas, María J; Bresciani, Guilherme; González-Gallego, Javier; Fighera, Michele Rechia; Royes, Luiz Fernando Freire

2017-09-01

An early inflammatory response and oxidative stress are implicated in the signal transduction that alters both hepatic redox status and mitochondrial function after traumatic brain injury (TBI). Peripheral oxidative/inflammatory responses contribute to neuronal dysfunction after TBI Exercise training alters the profile of oxidative-inflammatory status in liver and protects against acute hyperglycaemia and a cerebral inflammatory response after TBI. Approaches such as exercise training, which attenuates neuronal damage after TBI, may have therapeutic potential through modulation of responses by metabolic organs. The vulnerability of the body to oxidative/inflammatory in TBI is significantly enhanced in sedentary compared to physically active counterparts. Although systemic responses have been described after traumatic brain injury (TBI), little is known regarding potential interactions between brain and peripheral organs after neuronal injury. Accordingly, we aimed to investigate whether a peripheral oxidative/inflammatory response contributes to neuronal dysfunction after TBI, as well as the prophylactic role of exercise training. Animals were submitted to fluid percussion injury after 6 weeks of swimming training. Previous exercise training increased mRNA expression of X receptor alpha and ATP-binding cassette transporter, and decreased inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α and interleukin (IL)-6 expression per se in liver. Interestingly, exercise training protected against hepatic inflammation (COX-2, iNOS, TNF-α and IL-6), oxidative stress (decreases in non-protein sulfhydryl and glutathione, as well as increases in 2',7'-dichlorofluorescein diacetate oxidation and protein carbonyl), which altered hepatic redox status (increases in myeloperoxidase and superoxide dismutase activity, as well as inhibition of catalase activity) mitochondrial function (decreases in methyl-tetrazolium and Δψ, as well as inhibition of citrate synthase activity) and ion gradient homeostasis (inhibition of Na + ,K + -ATPase activity inhibition) when analysed 24 h after TBI. Previous exercise training also protected against dysglycaemia, impaired hepatic signalling (increase in phosphorylated c-Jun NH2-terminal kinase, phosphorylated decreases in insulin receptor substrate and phosphorylated AKT expression), high levels of circulating and neuronal cytokines, the opening of the blood-brain barrier, neutrophil infiltration and Na + ,K + -ATPase activity inhibition in the ipsilateral cortex after TBI. Moreover, the impairment of protein function, neurobehavioural (neuromotor dysfunction and spatial learning) disability and hippocampal cell damage in sedentary rats suggests that exercise training also modulates peripheral oxidative/inflammatory pathways in TBI, which corroborates the ever increasing evidence regarding health-related outcomes with respect to a physically active lifestyle. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Docosahexaenoic Acid Protects from Dendritic Pathology in an Alzheimer’s Disease Mouse Model

PubMed Central

Calon, Frédéric; Lim, Giselle P.; Yang, Fusheng; Morihara, Takashi; Teter, Bruce; Ubeda, Oliver; Rostaing, Phillippe; Triller, Antoine; Salem, Norman; Ashe, Karen H.; Frautschy, Sally A.; Cole, Greg M.

2005-01-01

Learning and memory depend on dendritic spine actin assembly and docosahexaenoic acid (DHA), an essential n-3 (omega-3) polyunsaturated fatty acid (PFA). High DHA consumption is associated with reduced Alzheimer’s disease (AD) risk, yet mechanisms and therapeutic potential remain elusive. Here, we report that reduction of dietary n-3 PFA in an AD mouse model resulted in 80%–90% losses of the p85α subunit of phosphatidylinositol 3-kinase and the postsynaptic actin-regulating protein drebrin, as in AD brain. The loss of postsynaptic proteins was associated with increased oxidation, without concomitant neuron or pre-synaptic protein loss. N-3 PFA depletion increased caspase-cleaved actin, which was localized in dendrites ultrastructurally. Treatment of n-3 PFA-restricted mice with DHA protected against these effects and behavioral deficits and increased antiapoptotic BAD phosphorylation. Since n-3 PFAs are essential for p85-mediated CNS insulin signaling and selective protection of postsynaptic proteins, these findings have implications for neurodegenerative diseases where synaptic loss is critical, especially AD. PMID:15339646

Structure of the cobalamin-binding protein of a putative O-demethylase from Desulfitobacterium hafniense DCB-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sjuts, Hanno; Dunstan, Mark S.; Fisher, Karl

2013-08-01

The first crystal structure of the vitamin B12-binding protein from a three-component O-demethylase enzyme system is reported. During O-demethylation methyl groups are transferred from phenyl methyl ethers to tetrahydrofolate via methyl-B12 intermediates. This study describes the identification and the structural and spectroscopic analysis of a cobalamin-binding protein (termed CobDH) implicated in O-demethylation by the organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2. The 1.5 Å resolution crystal structure of CobDH is presented in the cobalamin-bound state and reveals that the protein is composed of an N-terminal helix-bundle domain and a C-terminal Rossmann-fold domain, with the cobalamin coordinated in the base-off/His-on conformation similar tomore » other cobalamin-binding domains that catalyse methyl-transfer reactions. EPR spectroscopy of CobDH confirms cobalamin binding and reveals the presence of a cob(III)alamin superoxide, indicating binding of oxygen to the fully oxidized cofactor. These data provide the first structural insights into the methyltransferase reactions that occur during O-demethylation by D. hafniense.« less

Regulation of protein metabolism by glutamine: implications for nutrition and health.

PubMed

Xi, Pengbin; Jiang, Zongyong; Zheng, Chuntian; Lin, Yingcai; Wu, Guoyao

2011-01-01

Glutamine is the most abundant free alpha-amino acid in plasma and skeletal muscle. This nutrient plays an important role in regulating gene expression, protein turnover, anti-oxidative function, nutrient metabolism, immunity, and acid-base balance. Interestingly, intracellular and extracellular concentrations of glutamine exhibit marked reductions in response to infection, sepsis, severe burn, cancer, and other pathological factors. This raised an important question of whether glutamine may be a key mediator of muscle loss and negative nitrogen balance in critically ill and injured patients. Therefore, since the initial reports in late 1980s that glutamine could stimulate protein synthesis and inhibit proteolysis in rat skeletal muscle, there has been growing interest in the use of this functional amino acid to improve protein balance under various physiological and disease conditions. Although inconsistent results have appeared in the literature regarding a therapeutic role of glutamine in clinical medicine, a majority of studies indicate that supplementing appropriate doses of glutamine to enteral diets or parenteral solutions is beneficial for improving nitrogen balance in animals or humans with glutamine deficiency.

Lysinuric protein intolerance (LPI): a multi organ disease by far more complex than a classic urea cycle disorder.

PubMed

Ogier de Baulny, Hélène; Schiff, Manuel; Dionisi-Vici, Carlo

2012-05-01

Lysinuric protein intolerance (LPI) is an inherited defect of cationic amino acid (lysine, arginine and ornithine) transport at the basolateral membrane of intestinal and renal tubular cells caused by mutations in SLC7A7 encoding the y(+)LAT1 protein. LPI has long been considered a relatively benign urea cycle disease, when appropriately treated with low-protein diet and l-citrulline supplementation. However, the severe clinical course of this disorder suggests that LPI should be regarded as a severe multisystem disease with uncertain outcome. Specifically, immune dysfunction potentially attributable to nitric oxide (NO) overproduction secondary to arginine intracellular trapping (due to defective efflux from the cell) might be a crucial pathophysiological route explaining many of LPI complications. The latter comprise severe lung disease with pulmonary alveolar proteinosis, renal disease, hemophagocytic lymphohistiocytosis with subsequent activation of macrophages, various auto-immune disorders and an incompletely characterized immune deficiency. These results have several therapeutic implications, among which lowering the l-citrulline dosage may be crucial, as excessive citrulline may worsen intracellular arginine accumulation. Copyright © 2012 Elsevier Inc. All rights reserved.

Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

PubMed Central

Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

2014-01-01

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

PubMed

Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

2014-02-01

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

Simultaneous detection of changes in protein expression and oxidative modification as function of age and APOE genotype in human APOE-targeted replacement mice

EPA Science Inventory

Background The purpose of this study was to improve the current method for detecting differentially-oxidized (carbonyl-modified) proteins by 2D-DIGE, while at the same time determining changes in total protein expression. Protein oxidation is a widely accepted model of aging and...

An Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

PubMed Central

McClintock, Carlee S; Hettich, Robert L.

2012-01-01

Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent – hydroxyl radicals – for these measurements; however, these approaches range significantly in their complexity and expense of operation. This research expands upon earlier work to enhance the controllability of boron-doped diamond (BDD) electrochemistry as an easily accessible tool for producing hydroxyl radicals in order to oxidize a range of intact proteins. Efforts to modulate oxidation level while minimizing the adsorption of protein to the electrode involved the use of relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber. Additionally, a different cell activation approach using variable voltage to supply a controlled current allowed us to precisely tune the extent of oxidation in a protein-dependent manner. In order to gain perspective on the level of protein adsorption onto the electrode surface, studies were conducted to monitor protein concentration during electrolysis and gauge changes in the electrode surface between cell activation events. This report demonstrates the successful use of BDD electrochemistry for greater precision in generating a target number of oxidation events upon intact proteins. PMID:23210708

Pulsed Electron Beam Water Radiolysis for Sub-Microsecond Hydroxyl Radical Protein Footprinting

PubMed Central

Watson, Caroline; Janik, Ireneusz; Zhuang, Tiandi; Charvátová, Olga; Woods, Robert J.; Sharp, Joshua S.

2009-01-01

Hydroxyl radical footprinting is a valuable technique for studying protein structure, but care must be taken to ensure that the protein does not unfold during the labeling process due to oxidative damage. Footprinting methods based on sub-microsecond laser photolysis of peroxide that complete the labeling process faster than the protein can unfold have been recently described; however, the mere presence of large amounts of hydrogen peroxide can also cause uncontrolled oxidation and minor conformational changes. We have developed a novel method for sub-microsecond hydroxyl radical protein footprinting using a pulsed electron beam from a 2 MeV Van de Graaff electron accelerator to generate a high concentration of hydroxyl radicals by radiolysis of water. The amount of oxidation can be controlled by buffer composition, pulsewidth, dose, and dissolved nitrous oxide gas in the sample. Our results with ubiquitin and β-lactoglobulin A demonstrate that one sub-microsecond electron beam pulse produces extensive protein surface modifications. Highly reactive residues that are buried within the protein structure are not oxidized, indicating that the protein retains its folded structure during the labeling process. Time-resolved spectroscopy indicates that the major part of protein oxidation is complete in a timescale shorter than that of large scale protein motions. PMID:19265387

Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress–mediated apoptosis: possible implication in asthma

PubMed Central

Merendino, Anna M.; Paul, Catherine; Vignola, Antonio M.; Costa, Maria A.; Melis, Mario; Chiappara, Giuseppina; Izzo, V.; Bousquet, J.; Arrigo, André-Patrick

2002-01-01

Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide–induced apoptosis in human bronchial epithelial 16–HBE cells when they were genetically modified to express reduced levels of Hsp27. PMID:12482203

Insulin restores neuronal nitric oxide synthase expression and function that is lost in diabetic gastropathy

PubMed Central

Watkins, Crystal C.; Sawa, Akira; Jaffrey, Samie; Blackshaw, Seth; Barrow, Roxanne K.; Snyder, Solomon H.; Ferris, Christopher D.

2000-01-01

Gastrointestinal dysfunction is common in diabetic patients. In genetic (nonobese diabetic) and toxin-elicited (streptozotocin) models of diabetes in mice, we demonstrate defects in gastric emptying and nonadrenergic, noncholinergic relaxation of pyloric muscle, which resemble defects in mice harboring a deletion of the neuronal nitric oxide synthase gene (nNOS). The diabetic mice manifest pronounced reduction in pyloric nNOS protein and mRNA. The decline of nNOS in diabetic mice does not result from loss of myenteric neurons. nNOS expression and pyloric function are restored to normal levels by insulin treatment. Thus diabetic gastropathy in mice reflects an insulin-sensitive reversible loss of nNOS. In diabetic animals, delayed gastric emptying can be reversed with a phosphodiesterase inhibitor, sildenafil. These findings have implications for novel therapeutic approaches and may clarify the etiology of diabetic gastropathy. PMID:10930440

Proanthocyanidins against Oxidative Stress: From Molecular Mechanisms to Clinical Applications

PubMed Central

Xiong, Xia; Lai, Rui

2018-01-01

Proanthocyanidins (PCs) are naturally occurring polyphenolic compounds abundant in many vegetables, plant skins (rind/bark), seeds, flowers, fruits, and nuts. Numerous in vitro and in vivo studies have demonstrated myriad effects potentially beneficial to human health, such as antioxidation, anti-inflammation, immunomodulation, DNA repair, and antitumor activity. Accumulation of prooxidants such as reactive oxygen species (ROS) exceeding cellular antioxidant capacity results in oxidative stress (OS), which can damage macromolecules (DNA, lipids, and proteins), organelles (membranes and mitochondria), and whole tissues. OS is implicated in the pathogenesis and exacerbation of many cardiovascular, neurodegenerative, dermatological, and metabolic diseases, both through direct molecular damage and secondary activation of stress-associated signaling pathways. PCs are promising natural agents to safely prevent acute damage and control chronic diseases at relatively low cost. In this review, we summarize the molecules and signaling pathways involved in OS and the corresponding therapeutic mechanisms of PCs. PMID:29750172

Scleroderma Autoantigens Are Uniquely Fragmented by Metal-catalyzed Oxidation Reactions: Implications for Pathogenesis

PubMed Central

Casciola-Rosen, Livia; Wigley, Fredrick; Rosen, Antony

1997-01-01

The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity. The reversible ischemia reperfusion which characterizes scleroderma has focused attention on reactive oxygen species as molecules which might induce autoantigen fragmentation. We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner. Multiple features of the fragmentation reaction and its inhibition indicate that these autoantigens possess metal-binding sites, which focus metal-catalyzed oxidation reactions (and consequent fragmentation) to specific regions of the antigens. These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease. PMID:8996243

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Myeongsang; Baek, Inchul; Choi, Hyunsung

Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aβ-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau proteinmore » structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures. - Highlights: • Lysine mutation effect alters the structure conformation and characteristic of tau. • Over the 15 layers both WT and Q2K models, both tau proteins undergo fractions. • Lysine mutation causes the increment of non-bonded energy and solvent accessible surface area. • Structural instability of Q2K model was proved by the number of hydrogen bonds analysis.« less

Study of safety implications for shuttle launched spacecraft using fluorinated oxidizers. Volume 2: Executive summary

NASA Technical Reports Server (NTRS)

1975-01-01

An abbreviated version of the conclusions dealing with the safety implications of using liquid fluorinated oxidizers on space shuttle launched spacecraft was presented. The complete version was presented in volume 1.

Thiol oxidation and di-tyrosine formation in human plasma proteins induced by inflammatory concentrations of hypochlorous acid.

PubMed

Colombo, Graziano; Clerici, Marco; Altomare, Alessandra; Rusconi, Francesco; Giustarini, Daniela; Portinaro, Nicola; Garavaglia, Maria Lisa; Rossi, Ranieri; Dalle-Donne, Isabella; Milzani, Aldo

2017-01-30

In this study, we assessed the oxidative damage occurring in plasma proteins when human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). We used specific thiol labelling and Western blot analyses to determine protein thiol oxidation, as well as analytical gel filtration HPLC coupled to fluorescence detection to explore formation of high molecular weight (HMW) protein aggregates. Thiol-containing proteins oxidized by HOCl were identified by redox proteomics. Mass spectrometry (MS) analysis was performed to elucidate the protein composition of HMW aggregates. α1-antitrypsin, transthyretin, and haptoglobin showed thiol oxidation at HOCl concentrations higher than those causing complete oxidation of albumin. At the highest HOCl concentrations, formation of carbonylated and di-tyrosine cross-linked HMW protein aggregates also occurred. MS analysis identified fibrinogen, complement C3 and apolipoprotein A-I as components of HMW protein aggregates. These results could be relevant for human diseases characterized by inflammatory conditions in which myeloperoxidase and HOCl are involved. In this study we evaluated the oxidative damage occurring on plasma proteins when reconstituted human blood was exposed to inflammatory concentrations of hypochlorous acid (HOCl). Pathophysiological concentrations of HOCl are able to induce different modifications on plasma proteins such as carbonylation, sulfhydryl oxidation and formation of high molecular weight (HMW) protein aggregates characterized by di-tyrosine fluorescence. There are two relevant aspects emerging from this paper. The first one consists on identifying low abundant proteins undergoing sulfhydryl oxidation by biotin-maleimide derivatization followed by MALDI-TOF mass spectrometry. This approach suggests three low-abundant proteins undergoing HOCl-induced oxidation: transthyretin, α1-antitrypsin, and haptoglobin. In addition, we analysed HMW protein aggregates forming after HOCl exposure. These aggregates are characterized by carbonylation, intra- and/or intermolecular di-tyrosine bridges. After their isolation from SDS-PAGE gel electrophoresis, using electrospray tandem mass spectrometry coupled to reversed-phase nanoscale capillary liquid chromatography, we identified some protein constituents of these HMW aggregates such as α, β, γ fibrinogen chains, apolipoprotein A-I and complement C3. In particular, our work highlights how fibrinogen is an important constituent of HOCl-induced HMW protein aggregates validating the mass spectrometry result with additional experiments. Further investigations are required in order to evaluate the possibility to use carbonylated and di-Tyr cross-linked HMW protein aggregates as (early) biomarkers for disease progression in inflammatory conditions in which myeloperoxidase and HOCl are involved. Copyright © 2016 Elsevier B.V. All rights reserved.

Combined acute hyperglycemic and hyperinsulinemic clamp induced profibrotic and proinflammatory responses in the kidney

PubMed Central

DeSilva, Kristin; Sorice, Gian Pio; Muscogiuri, Giovanna; Jimenez, Fabio; Ahuja, Seema; Barnes, Jefferey L.; Choudhury, Goutam Ghosh; Musi, Nicolas; DeFronzo, Ralph; Kasinath, Balakuntalam S.

2013-01-01

Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate whether short duration of combined hyperglycemia and hyperinsulinemia, mimicking the metabolic environment of prediabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n = 4) or 7 h of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n = 6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical transforming growth factor-β (TGF-β) and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mammalian target of rapamycin (mTOR) complexes, and ERK signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of Toll-like receptor 4 (TLR4) and infiltration of CD68-positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation, and oxidative stress in the kidney. This finding could have implications for the control of short-term rises in blood glucose in diabetic individuals at risk of developing kidney disease. PMID:24108867

Combined acute hyperglycemic and hyperinsulinemic clamp induced profibrotic and proinflammatory responses in the kidney.

PubMed

Mariappan, Meenalakshmi M; DeSilva, Kristin; Sorice, Gian Pio; Muscogiuri, Giovanna; Jimenez, Fabio; Ahuja, Seema; Barnes, Jefferey L; Choudhury, Goutam Ghosh; Musi, Nicolas; DeFronzo, Ralph; Kasinath, Balakuntalam S

2014-02-01

Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate whether short duration of combined hyperglycemia and hyperinsulinemia, mimicking the metabolic environment of prediabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n = 4) or 7 h of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n = 6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical transforming growth factor-β (TGF-β) and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mammalian target of rapamycin (mTOR) complexes, and ERK signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of Toll-like receptor 4 (TLR4) and infiltration of CD68-positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation, and oxidative stress in the kidney. This finding could have implications for the control of short-term rises in blood glucose in diabetic individuals at risk of developing kidney disease.

Osteoporosis and alzheimer pathology: Role of cellular stress response and hormetic redox signaling in aging and bone remodeling

PubMed Central

Cornelius, Carolin; Koverech, Guido; Crupi, Rosalia; Di Paola, Rosanna; Koverech, Angela; Lodato, Francesca; Scuto, Maria; Salinaro, Angela T.; Cuzzocrea, Salvatore; Calabrese, Edward J.; Calabrese, Vittorio

2014-01-01

Alzheimer’s disease (AD) and osteoporosis are multifactorial progressive degenerative disorders. Increasing evidence shows that osteoporosis and hip fracture are common complication observed in AD patients, although the mechanisms underlying this association remain poorly understood. Reactive oxygen species (ROS) are emerging as intracellular redox signaling molecules involved in the regulation of bone metabolism, including receptor activator of nuclear factor-κB ligand-dependent osteoclast differentiation, but they also have cytotoxic effects that include lipoperoxidation and oxidative damage to proteins and DNA. ROS generation, which is implicated in the regulation of cellular stress response mechanisms, is an integrated, highly regulated, process under control of redox sensitive genes coding for redox proteins called vitagenes. Vitagenes, encoding for proteins such as heat shock proteins (Hsps) Hsp32, Hsp70, the thioredoxin, and the sirtuin protein, represent a systems controlling a complex network of intracellular signaling pathways relevant to life span and involved in the preservation of cellular homeostasis under stress conditions. Consistently, nutritional anti-oxidants have demonstrated their neuroprotective potential through a hormetic-dependent activation of vitagenes. The biological relevance of dose–response affects those strategies pointing to the optimal dosing to patients in the treatment of numerous diseases. Thus, the heat shock response has become an important hormetic target for novel cytoprotective strategies focusing on the pharmacological development of compounds capable of modulating stress response mechanisms. Here we discuss possible signaling mechanisms involved in the activation of vitagenes which, relevant to bone remodeling and through enhancement of cellular stress resistance provide a rationale to limit the deleterious consequences associated to homeostasis disruption with consequent impact on the aging process. PMID:24959146

Molecular modeling, dynamics studies and density functional theory approaches to identify potential inhibitors of SIRT4 protein from Homo sapiens : a novel target for the treatment of type 2 diabetes.

PubMed

Choubey, Sanjay K; Prabhu, Dhamodharan; Nachiappan, Mutharasappan; Biswal, Jayshree; Jeyakanthan, Jeyaraman

2017-11-01

Type 2 diabetes is one of the biggest health challenges in the world and WHO projects it to be the 7th leading cause of death in 2030. It is a chronic condition affecting the way our body metabolizes sugar. Insulin resistance is high risk factor marked by expression of Lipoprotein Lipases and Peroxisome Proliferator-Activated Receptor that predisposes to type 2 diabetes. AMP-dependent protein kinase in AMPK signaling pathway is a central sensor of energy status. Deregulation of AMPK signaling leads to inflammation, oxidative stress, and deactivation of autophagy which are implicated in pathogenesis of insulin resistance. SIRT4 protein deactivates AMPK as well as directly inhibits insulin secretion. SIRT4 overexpression leads to dyslipidimeia, decreased fatty acid oxidation, and lipogenesis which are the characteristic features of insulin resistance promoting type 2 diabetes. This makes SIRT4 a novel therapeutic target to control type 2 diabetes. Virtual screening and molecular docking studies were performed to obtain potential ligands. To further optimize the geometry of protein-ligand complexes Quantum Polarized Ligand Docking was performed. Binding Free Energy was calculated for the top three ligand molecules. In view of exploring the stereoelectronic features of the ligand, density functional theory approach was implemented at B3LYP/6-31G* level. 30 ns MD simulation studies of the protein-ligand complexes were done. The present research work proposes ZINC12421989 as potential inhibitor of SIRT4 with docking score (-7.54 kcal/mol), docking energy (-51.34 kcal/mol), binding free energy (-70.21 kcal/mol), and comparatively low energy gap (-0.1786 eV) for HOMO and LUMO indicating reactivity of the lead molecule.

Reactive Oxygen Species-Mediated Loss of Synaptic Akt1 Signaling Leads to Deficient Activity-Dependent Protein Translation Early in Alzheimer's Disease

PubMed Central

Ahmad, Faraz; Singh, Kunal; Das, Debajyoti; Gowaikar, Ruturaj; Shaw, Eisha; Ramachandran, Arathy; Rupanagudi, Khader Valli; Kommaddi, Reddy Peera; Bennett, David A.

2017-01-01

Abstract Aims: Synaptic deficits are known to underlie the cognitive dysfunction seen in Alzheimer's disease (AD). Generation of reactive oxygen species (ROS) by β-amyloid has also been implicated in AD pathogenesis. However, it is unclear whether ROS contributes to synaptic dysfunction seen in AD pathogenesis and, therefore, we examined whether altered redox signaling could contribute to synaptic deficits in AD. Results: Activity dependent but not basal translation was impaired in synaptoneurosomes from 1-month old presymptomatic APPSwe/PS1ΔE9 (APP/PS1) mice, and this deficit was sustained till middle age (MA, 9–10 months). ROS generation leads to oxidative modification of Akt1 in the synapse and consequent reduction in Akt1-mechanistic target of rapamycin (mTOR) signaling, leading to deficiency in activity-dependent protein translation. Moreover, we found a similar loss of activity-dependent protein translation in synaptoneurosomes from postmortem AD brains. Innovation: Loss of activity-dependent protein translation occurs presymptomatically early in the pathogenesis of AD. This is caused by ROS-mediated loss of pAkt1, leading to reduced synaptic Akt1-mTOR signaling and is rescued by overexpression of Akt1. ROS-mediated damage is restricted to the synaptosomes, indicating selectivity. Conclusions: We demonstrate that ROS-mediated oxidative modification of Akt1 contributes to synaptic dysfunction in AD, seen as loss of activity-dependent protein translation that is essential for synaptic plasticity and maintenance. Therapeutic strategies promoting Akt1-mTOR signaling at synapses may provide novel target(s) for disease-modifying therapy in AD. Antioxid. Redox Signal. 27, 1269–1280. PMID:28264587

Protein oxidation and proteolysis during roasting and in vitro digestion of fish (Acipenser gueldenstaedtii).

PubMed

Hu, Lyulin; Ren, Sijie; Shen, Qing; Ye, Xingqian; Chen, Jianchu; Ling, Jiangang

2018-04-15

Roasted fish enjoys great popularity in Asia, but how roasting and subsequent digestion influence the oxidation and proteolysis of fish meat is unknown. This paper is aimed to investigate the effect of roasting time on lipid and protein oxidation and their evolution and consequence on proteolysis during simulated digestion of fish fillets. Several oxidation markers (TBARS, free thiols, total carbonyls and Schiff bases) were employed to assess the oxidation of fish. SDS-PAGE and TBNS assay for free amino groups were used to study the proteolysis during gastrointestinal digestion. The results showed that significant lipid and protein oxidative changes occurring in roasted fish fillets were reinforced after gastric digestion and were much more intense after intestinal digestion. Throughout the roasting and digestion, close interconnection between lipid and protein was also manifested as the levels of total carbonyls and Schiff bases rose while TBARS fell. Furthermore, free amino groups decreased with prolonged roasting time, signifying protein oxidation before digestion resulted in impaired proteolysis during digestion. This paper indicated the lipid and protein oxidation of fish fillets could be dependent on time of roasting, and the oxidation continued to develop and have an impact on proteolysis during in vitro digestion. This article is protected by copyright. All rights reserved.

Exosome-derived microRNAs in cancer metabolism: possible implications in cancer diagnostics and therapy.

PubMed

Tomasetti, Marco; Lee, Wan; Santarelli, Lory; Neuzil, Jiri

2017-01-20

Malignant progression is greatly affected by dynamic cross-talk between stromal and cancer cells. Exosomes are secreted nanovesicles that have key roles in cell-cell communication by transferring nucleic acids and proteins to target cells and tissues. Recently, MicroRNAs (miRs) and their delivery in exosomes have been implicated in physiological and pathological processes. Tumor-delivered miRs, interacting with stromal cells in the tumor microenvironment, modulate tumor progression, angiogenesis, metastasis and immune escape. Altered cell metabolism is one of the hallmarks of cancer. A number of different types of tumor rely on mitochondrial metabolism by triggering adaptive mechanisms to optimize their oxidative phosphorylation in relation to their substrate supply and energy demands. Exogenous exosomes can induce metabolic reprogramming by restoring the respiration of cancer cells and supress tumor growth. The exosomal miRs involved in the modulation of cancer metabolism may be potentially utilized for better diagnostics and therapy.

Albumin in chronic liver disease: structure, functions and therapeutic implications.

PubMed

Spinella, Rosaria; Sawhney, Rohit; Jalan, Rajiv

2016-01-01

Human serum albumin is a critical plasma protein produced by the liver with a number of accepted clinical indications in chronic liver disease including management of circulatory and renal dysfunction in patients with ascites. Advanced cirrhosis is characterised by reduced albumin concentration as well as impaired albumin function as a result of specific structural changes and oxidative damage. Traditionally, the biologic and therapeutic role of albumin in liver disease was attributed to its oncotic effects but it is now understood that albumin has a wide range of other important physiologic functions such as immunomodulation, endothelial stabilisation, antioxidant effects and binding multiple drugs, toxins and other molecules. This review discusses the multifunctional properties of albumin and, in particular, the biologic and clinical implications of structural and functional changes of albumin that are associated with cirrhosis. Based on these insights, we explore the current and potential future therapeutic uses of albumin in liver disease.

Oxidatively denatured proteins are degraded by an ATP-independent proteolytic pathway in Escherichia coli.

PubMed

Davies, K J; Lin, S W

1988-01-01

E. coli contains a soluble proteolytic pathway which can recognize and degrade oxidatively denatured proteins and protein fragments, and which may act as a "secondary antioxidant defense." We now provide evidence that this proteolytic pathway is distinct from the previously described ATP-dependent, and protease "La"-dependent, pathway which may degrade other abnormal proteins. Cells (K12) which were depleted of ATP, by arsenate treatment or anaerobic incubation (after growth on succinate), exhibited proteolytic responses to oxidative stress which were indistinguishable from those observed in cells with normal ATP levels. Furthermore, the proteolytic responses to oxidative damage by menadione or H2O2 were almost identical in the isogenic strains RM312 (a K12 derivative) and RM1385 (a lon deletion mutant of RM312). Since the lon (or capR) gene codes for the ATP-dependent protease "La," these results indicate that neither ATP nor protease "La" are required for the degradation of oxidatively denatured proteins. We next prepared cell-free extracts of K12, RM312, and RM1385 and tested the activity of their soluble proteases against proteins (albumin, hemoglobin, superoxide dismutase, catalase) which had been oxidatively denatured (in vitro) by exposure to .OH, .OH + O2- (+O2), H2O2, or ascorbate plus iron. The breakdown of oxidatively denatured proteins was several-fold higher than that of untreated proteins in extracts from all three strains, and ATP did not stimulate degradation. Incubation of extracts at 45 degrees C, which inactivates protease "La," actually stimulated the degradation of oxidatively denatured proteins. Although Ca2+ had little effect on proteolysis, serine reagents, transition metal chelators, and hemin effectively inhibited the degradation of oxidatively denatured proteins in both intact cells and cell-free extracts. Degradation of oxidatively denatured proteins in cell-free extracts was maximal at pH 7.8, and was unaffected by dialysis of the extracts against membranes with molecular weight cutoffs as high as 50,000. Our results indicate the presence of a neutral, ATP- and calcium- independent proteolytic pathway in the E. coli cytosol, which contains serine- and metallo- proteases (with molecular weights greater than 50,000), and which preferentially degrades oxidatively denatured proteins.

Jumonji Domain Containing Protein 6: A Novel Oxygen Sensor in the Human Placenta.

PubMed

Alahari, Sruthi; Post, Martin; Caniggia, Isabella

2015-08-01

Persistent low oxygen is implicated in the pathogenesis of placental-associated pathologies such as preeclampsia, a serious disorder of pregnancy. Emerging evidence implicates a novel family of Jumonji C catalytic domain proteins as mediators of hypoxic gene expression. Here, we investigated the regulatory relationship between Jumonji C domain containing protein 6 (JMJD6) and hypoxia-inducible factor (HIF)1A in the human placenta at physiological and pathological conditions. JMJD6 expression inversely correlated with changes in oxygen tension during early placental development, ie, high at 7-9 weeks when-partial pressure of O2 is low and declining afterwards when-partial pressure of O2 increases. Moreover, JMJD6 protein was significantly elevated in early-onset preeclamptic placentae, localizing to the syncytiotrophoblast layer and syncytial knots. Exposure of primary isolated trophoblast cells, human villous explants, and JEG3 choriocarcinoma cells to low oxygen (3%) and sodium nitroprusside (inducer of oxidative stress) also resulted in elevated JMJD6 levels, which was abrogated by HIF1A knockdown. In normoxia, knockdown of JMJD6 in JEG3 cells stabilized HIF1A with a concomitant decrease in von Hippel-Lindau (VHL) tumor suppressor protein, a negative regulator of HIF1A stability. In contrast, overexpression of JMJD6 enhanced VHL expression and destabilized HIF1A. JMJD6 regulation of VHL stability did not involve the ubiquitin-proteasome system but likely occurred through lysyl hydroxylation and small ubiquitin-like modifier 1-dependent small ubiquitin-like modifierylation. In summary, our data signify a novel role for JMJD6 as an oxygen sensor in the human placenta, and alterations in the JMJD6-VHL-HIF1A feedback loop may indirectly contribute to elevated HIF1A found in preeclampsia.

Structural and functional characterisation of multi-copper oxidase CueO from lignin-degrading bacterium Ochrobactrum sp. reveal its activity towards lignin model compounds and lignosulfonate.

PubMed

Granja-Travez, Rommel Santiago; Wilkinson, Rachael C; Persinoti, Gabriela Felix; Squina, Fabio M; Fülöp, Vilmos; Bugg, Timothy D H

2018-05-01

The identification of enzymes responsible for oxidation of lignin in lignin-degrading bacteria is of interest for biotechnological valorization of lignin to renewable chemical products. The genome sequences of two lignin-degrading bacteria, Ochrobactrum sp., and Paenibacillus sp., contain no B-type DyP peroxidases implicated in lignin degradation in other bacteria, but contain putative multicopper oxidase genes. Multi-copper oxidase CueO from Ochrobactrum sp. was expressed and reconstituted as a recombinant laccase-like enzyme, and kinetically characterized. Ochrobactrum CueO shows activity for oxidation of β-aryl ether and biphenyl lignin dimer model compounds, generating oxidized dimeric products, and shows activity for oxidation of Ca-lignosulfonate, generating vanillic acid as a low molecular weight product. The crystal structure of Ochrobactrum CueO (OcCueO) has been determined at 1.1 Å resolution (PDB: 6EVG), showing a four-coordinate mononuclear type I copper center with ligands His495, His434 and Cys490 with Met500 as an axial ligand, similar to that of Escherichia coli CueO and bacterial azurin proteins, whereas fungal laccase enzymes contain a three-coordinate type I copper metal center. A trinuclear type 2/3 copper cluster was modeled into the active site, showing similar structure to E. coli CueO and fungal laccases, and three solvent channels leading to the active site. Site-directed mutagenesis was carried out on amino acid residues found in the solvent channels, indicating the importance for residues Asp102, Gly103, Arg221, Arg223, and Asp462 for catalytic activity. The work identifies a new bacterial multicopper enzyme with activity for lignin oxidation, and implicates a role for bacterial laccase-like multicopper oxidases in some lignin-degrading bacteria. Structural data are available in the PDB under the accession number 6EVG. © 2018 Federation of European Biochemical Societies.

Thiazolidine-2,4-dione derivatives: programmed chemical weapons for key protein targets of various pathological conditions.

PubMed

Chadha, Navriti; Bahia, Malkeet Singh; Kaur, Maninder; Silakari, Om

2015-07-01

Thiazolidine-2,4-dione is an extensively explored heterocyclic nucleus for designing of novel agents implicated for a wide variety of pathophysiological conditions, that is, diabetes, diabetic complications, cancer, arthritis, inflammation, microbial infection, and melanoma, etc. The current paradigm of drug development has shifted to the structure-based drug design, since high-throughput screenings have continued to generate disappointing results. The gap between hit generation and drug establishment can be narrowed down by investigation of ligand interactions with its receptor protein. Therefore, it would always be highly beneficial to gain knowledge of molecular level interactions between specific protein target and developed ligands; since this information can be maneuvered to design new molecules with improved protein fitting. Thus, considering this aspect, we have corroborated the information about molecular (target) level implementations of thiazolidine-2,4-diones (TZD) derivatives having therapeutic implementations such as, but not limited to, anti-diabetic (glitazones), anti-cancer, anti-arthritic, anti-inflammatory, anti-oxidant and anti-microbial, etc. The structure based SAR of TZD derivatives for various protein targets would serve as a benchmark for the alteration of existing ligands to design new ones with better binding interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

The putative leucine sensor Sestrin2 is hyperphosphorylated by acute resistance exercise but not protein ingestion in human skeletal muscle.

PubMed

Zeng, Nina; D'Souza, Randall F; Sorrenson, Brie; Merry, Troy L; Barnett, Matthew P G; Mitchell, Cameron J; Cameron-Smith, David

2018-06-01

Dietary protein and resistance exercise (RE) are both potent stimuli of the mammalian target of rapamycin complex 1 (mTORC1). Sestrins1, 2, 3 are multifunctional proteins that regulate mTORC1, stimulate autophagy and alleviate oxidative stress. Of this family, Sestrin2 is a putative leucine sensor implicated in mTORC1 and AMP-dependent protein kinase (AMPK) regulation. There is currently no data examining the responsiveness of Sestrin2 to dietary protein ingestion, with or without RE. In Study 1, 16 males ingested either 10 or 20 g of milk protein concentrate (MPC) with muscle biopsies collected pre, 90 and 210 min post-beverage consumption. In Study 2, 20 males performed a bout of RE immediately followed by the consumption of 9 g of MPC or carbohydrate placebo. Analysis of Sestrins, AMPK and antioxidant responses was examined. Dietary protein ingestion did not result in Sestrin2 mobility shift. After RE, Sestrin2 phosphorylation state was significantly altered and was not further modified by post-exercise protein or carbohydrate ingestion. With RE, AMPK phosphorylation remained stable, while the mRNA expressions of several antioxidants were upregulated. Dietary protein ingestion did not affect the signalling by the family of Sestrins. With RE, Sestrin2 was hyperphosphorylated, with no further evidence of a relationship to AMPK signalling.

Control mechanisms in mitochondrial oxidative phosphorylation☆

PubMed Central

Hroudová, Jana; Fišar, Zdeněk

2013-01-01

Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5’- triphosphate production is regulated by many control mechanism–firstly by oxygen, substrate level, adenosine-5’-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by “second control mechanisms,” such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5’-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production. PMID:25206677

Control mechanisms in mitochondrial oxidative phosphorylation.

PubMed

Hroudová, Jana; Fišar, Zdeněk

2013-02-05

Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5'- triphosphate production is regulated by many control mechanism-firstly by oxygen, substrate level, adenosine-5'-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by "second control mechanisms," such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5'-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.

Mitochondrial thermogenesis and obesity.

PubMed

Gambert, Ségolène; Ricquier, Daniel

2007-11-01

Thermogenesis is activated at the expense of carbon molecules. Mitochondria play a dominant role in oxidation and parallel heat production since the recovery of oxidation energy is less than perfect. Recent data of mitochondriogenesis and mitochondrial thermogenesis may boost research into certain aspects of obesity. Recent studies have outlined the unexpected decreased thermogenesis that limits fat loss during prolonged food restriction. Activation of fat oxidation in skeletal muscle remains a strategy against fat accumulation, however. Certain adipose depots have the potential to promote thermogenesis, either using mitochondrial uncoupling protein or independently. Peroxisome proliferator-activated receptor gamma coactivators alpha and ss are important regulators of mitochondria thermogenesis. Brain mitochondria are involved in the control of refeeding after starvation. This dual action of mitochondria inform their role in thermogenesis and energy partitioning. The importance of thyroid hormones in mitochondria thermogenesis is also confirmed. The clinical and research implications of these findings are that the mechanisms inhibiting adaptive thermogenesis during diet restriction should be investigated. An important field of research is the contribution of transcriptional coactivators to adipocyte plasticity since adipocytes have an underestimated ability to oxidise fatty acids in addition to their role in triglyceride storage.

P53 alters the cytotoxicity and genotoxicity for oxidized graphene in human B-lymphoblastoid cells

NASA Astrophysics Data System (ADS)

Petibone, Dayton Matthew

Widespread use of oxidized graphene nanomaterials in industry, medicine, and consumer products raises concern about potential adverse impacts on human health. The p53 tumor suppressor protein is crucial to maintaining cellular and genetic stability to prevent carcinogenesis. Here, we show that oxygen functionalized graphene (f-G) absorption and p53 functional status correlate with cytotoxicity and genotoxicity in human B-lymphoblastoid cells. Trends in f-G absorption by were dose-dependent. Cells with functional p53 exposed to f-G arrested in G0/G1 phase of the cell cycle, suppressed f-G induced reactive oxygen species (ROS), and had elevated apoptosis. While compared to p53 competent cells, the p53 deficient cells exposed to f-G accumulated in S-phase of the cell cycle, had elevated ROS levels, and evaded apoptosis. The f-G genotoxicity was evident as increased loss-of-heterozygosity mutants independent of p53 status, and structural chromosome damage in p53 deficient cells. These findings have broad implications for the safety and efficacy of oxidized graphene nanomaterials in industrial, consumer products and biomedical applications.

Mitochondrial dysfunction precedes neurodegeneration in mahogunin (Mgrn1) mutant mice

PubMed Central

Sun, Kaihua; Johnson, Brian S.; Gunn, Teresa M.

2007-01-01

Oxidative stress, ubiquitination defects and mitochondrial dysfunction are commonly associated with neurodegeneration. Mice lacking mahogunin ring finger-1 (MGRN1) or attractin (ATRN) develop age-dependent spongiform neurodegeneration through an unknown mechanism. It has been suggested that they act in a common pathway. As MGRN1 is an E3 ubiquitin ligase, proteomic analysis of Mgrn1 mutant and control brains was performed to explore the hypothesis that loss of MGRN1 causes neurodegeneration via accumulation of its substrates. Many mitochondrial proteins were reduced in Mgrn1 mutants. Subsequent assays confirmed significantly reduced mitochondrial complex IV expression and activity as well as increased oxidative stress in mutant brains. Mitochondrial dysfunction was obvious many months before onset of vacuolation, implicating this as a causative factor. Compatible with the hypothesis that ATRN and MGRN1 act in the same pathway, mitochondrial dysfunction and increased oxidative stress were also observed in the brains of Atrn mutants. Our results suggest that the study of Mgrn1 and Atrn mutant mice will provide insight into a causative molecular mechanism common to many neurodegenerative disorders. PMID:17720281

Organ-Protective Effects of Red Wine Extract, Resveratrol, in Oxidative Stress-Mediated Reperfusion Injury

PubMed Central

Liu, Fu-Chao; Tsai, Hsin-I; Yu, Huang-Ping

2015-01-01

Resveratrol, a polyphenol extracted from red wine, possesses potential antioxidative and anti-inflammatory effects, including the reduction of free radicals and proinflammatory mediators overproduction, the alteration of the expression of adhesion molecules, and the inhibition of neutrophil function. A growing body of evidence indicates that resveratrol plays an important role in reducing organ damage following ischemia- and hemorrhage-induced reperfusion injury. Such protective phenomenon is reported to be implicated in decreasing the formation and reaction of reactive oxygen species and pro-nflammatory cytokines, as well as the mediation of a variety of intracellular signaling pathways, including the nitric oxide synthase, nicotinamide adenine dinucleotide phosphate oxidase, deacetylase sirtuin 1, mitogen-activated protein kinase, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, hemeoxygenase-1, and estrogen receptor-related pathways. Reperfusion injury is a complex pathophysiological process that involves multiple factors and pathways. The resveratrol is an effective reactive oxygen species scavenger that exhibits an antioxidative property. In this review, the organ-protective effects of resveratrol in oxidative stress-related reperfusion injury will be discussed. PMID:26161238

Ulinastatin activates haem oxygenase 1 antioxidant pathway and attenuates allergic inflammation

PubMed Central

Song, Dongmei; Song, Geng; Niu, Yinghao; Song, Wei; Wang, Jiantao; Yu, Lei; Yang, Jianwang; Lv, Xin; Steinberg, Harry; Liu, Shu Fang; Wang, Baoshan

2014-01-01

Background and Purpose Ulinastatin (UTI), a serine protease inhibitor, was recently found to have an anti-inflammatory action. However, the mechanisms mediating this anti-inflammatory effect are not well understood. This study tested the hypothesis that UTI suppresses allergic inflammation by inducing the expression of haem oxygenase 1 (HO1). Experimental Approach Control mice and mice sensitized (on days 1, 9 and 14) and challenged (on days 21 to 27) with ovalbumin (OVA) were treated with UTI. The effects of UTI on basal expression of HO1 and that induced by OVA challenge were examined. The involvement of UTI-induced HO1 expression in anti-inflammatory and antioxidant effects of UTI was also evaluated. Key Results UTI markedly increased basal HO1 protein expression in lungs of control mice in a time- and dose-dependent manner, and augmented HO1 protein expression induced by OVA. The up-regulation of HO1 mediated by UTI in sensitized and OVA-challenged mice was associated with reduced airway inflammation, alleviated tissue injury, reduced oxidant stress and enhanced antioxidant enzyme activities. Inhibition of HO1 activity using HO1 inhibitor, zinc protoporphyrin, attenuated inhibitory effects of UTI on inflammation and oxidant stress, and its stimulant effects on antioxidant enzyme activities. Mechanistic analysis showed that UTI increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), stimulated Nrf2 DNA binding activity and concomitantly up-regulated HO1 mRNA expression. Conclusions and Implications UTI is a potent and naturally occurring inducer of HO1 expression. HO1 up-regulation contributes significantly to the anti-inflammatory and organ-protective effects of UTI, which has important research and therapeutic implications. PMID:24835359

Discordant signaling and autophagy response to fasting in hearts of obese mice: Implications for ischemia tolerance

PubMed Central

Kooren, Joel A.; Parker, Sarah J.; Tucker, Kyle C.; Ravindran, Nandini; Ito, Bruce R.; Huang, Chengqun; Venkatraman, Vidya; Van Eyk, Jennifer E.; Gottlieb, Roberta A.; Mentzer, Robert M.

2016-01-01

Autophagy is regulated by nutrient and energy status and plays an adaptive role during nutrient deprivation and ischemic stress. Metabolic syndrome (MetS) is a hypernutritive state characterized by obesity, dyslipidemia, elevated fasting blood glucose levels, and insulin resistance. It has also been associated with impaired autophagic flux and larger-sized infarcts. We hypothesized that diet-induced obesity (DIO) affects nutrient sensing, explaining the observed cardiac impaired autophagy. We subjected male friend virus B NIH (FVBN) mice to a high-fat diet, which resulted in increased weight gain, fat deposition, hyperglycemia, insulin resistance, and larger infarcts after myocardial ischemia-reperfusion. Autophagic flux was impaired after 4 wk on a high-fat diet. To interrogate nutrient-sensing pathways, DIO mice were subjected to overnight fasting, and hearts were processed for biochemical and proteomic analysis. Obese mice failed to upregulate LC3-II or to clear p62/SQSTM1 after fasting, although mRNA for LC3B and p62/SQSTM1 were appropriately upregulated in both groups, demonstrating an intact transcriptional response to fasting. Energy- and nutrient-sensing signal transduction pathways [AMPK and mammalian target of rapamycin (mTOR)] also responded appropriately to fasting, although mTOR was more profoundly suppressed in obese mice. Proteomic quantitative analysis of the hearts under fed and fasted conditions revealed broad changes in protein networks involved in oxidative phosphorylation, autophagy, oxidative stress, protein homeostasis, and contractile machinery. In many instances, the fasting response was quite discordant between lean and DIO mice. Network analysis implicated the peroxisome proliferator-activated receptor and mTOR regulatory nodes. Hearts of obese mice exhibited impaired autophagy, altered proteome, and discordant response to nutrient deprivation. PMID:27199111

Discordant signaling and autophagy response to fasting in hearts of obese mice: Implications for ischemia tolerance.

PubMed

Andres, Allen M; Kooren, Joel A; Parker, Sarah J; Tucker, Kyle C; Ravindran, Nandini; Ito, Bruce R; Huang, Chengqun; Venkatraman, Vidya; Van Eyk, Jennifer E; Gottlieb, Roberta A; Mentzer, Robert M

2016-07-01

Autophagy is regulated by nutrient and energy status and plays an adaptive role during nutrient deprivation and ischemic stress. Metabolic syndrome (MetS) is a hypernutritive state characterized by obesity, dyslipidemia, elevated fasting blood glucose levels, and insulin resistance. It has also been associated with impaired autophagic flux and larger-sized infarcts. We hypothesized that diet-induced obesity (DIO) affects nutrient sensing, explaining the observed cardiac impaired autophagy. We subjected male friend virus B NIH (FVBN) mice to a high-fat diet, which resulted in increased weight gain, fat deposition, hyperglycemia, insulin resistance, and larger infarcts after myocardial ischemia-reperfusion. Autophagic flux was impaired after 4 wk on a high-fat diet. To interrogate nutrient-sensing pathways, DIO mice were subjected to overnight fasting, and hearts were processed for biochemical and proteomic analysis. Obese mice failed to upregulate LC3-II or to clear p62/SQSTM1 after fasting, although mRNA for LC3B and p62/SQSTM1 were appropriately upregulated in both groups, demonstrating an intact transcriptional response to fasting. Energy- and nutrient-sensing signal transduction pathways [AMPK and mammalian target of rapamycin (mTOR)] also responded appropriately to fasting, although mTOR was more profoundly suppressed in obese mice. Proteomic quantitative analysis of the hearts under fed and fasted conditions revealed broad changes in protein networks involved in oxidative phosphorylation, autophagy, oxidative stress, protein homeostasis, and contractile machinery. In many instances, the fasting response was quite discordant between lean and DIO mice. Network analysis implicated the peroxisome proliferator-activated receptor and mTOR regulatory nodes. Hearts of obese mice exhibited impaired autophagy, altered proteome, and discordant response to nutrient deprivation. Copyright © 2016 the American Physiological Society.

The Role of Nutrients in Protecting Mitochondrial Function and Neurotransmitter Signaling: Implications for the Treatment of Depression, PTSD, and Suicidal Behaviors.

PubMed

Du, Jing; Zhu, Ming; Bao, Hongkun; Li, Bai; Dong, Yilong; Xiao, Chunjie; Zhang, Grace Y; Henter, Ioline; Rudorfer, Matthew; Vitiello, Benedetto

2016-11-17

Numerous studies have linked severe stress to the development of major depressive disorder (MDD) and suicidal behaviors. Furthermore, recent preclinical studies from our laboratory and others have demonstrated that in rodents, chronic stress and the stress hormone cortisol cause oxidative damage to mitochondrial function and membrane lipids in the brain. Mitochondria play a key role in synaptic neurotransmitter signaling by providing adenosine triphosphate (ATP), mediating lipid and protein synthesis, buffering intracellular calcium, and regulating apoptotic and resilience pathways. Membrane lipids are similarly essential to central nervous system (CNS) function because cholesterol, polyunsaturated fatty acids, and sphingolipids form a lipid raft region, a special lipid region on the membrane that mediates neurotransmitter signaling through G-protein-coupled receptors and ion channels. Low serum cholesterol levels, low antioxidant capacity, and abnormal early morning cortisol levels are biomarkers consistently associated with both depression and suicidal behaviors. In this review, we summarize the manner in which nutrients can protect against oxidative damage to mitochondria and lipids in the neuronal circuits associated with cognitive and affective behaviors. These nutrients include ω3 fatty acids, antioxidants (vitamin C and zinc), members of the vitamin B family (Vitamin B12 and folic acid), and magnesium. Accumulating data have shown that these nutrients can enhance neurocognitive function, and may have therapeutic benefits for depression and suicidal behaviors. A growing body of studies suggests the intriguing possibility that regular consumption of these nutrients may help prevent the onset of mood disorders and suicidal behaviors in vulnerable individuals, or significantly augment the therapeutic effect of available antidepressants. These findings have important implications for the health of both military and civilian populations.

The Role of Nutrients in Protecting Mitochondrial Function and Neurotransmitter Signaling: Implications for the Treatment of Depression, PTSD, and Suicidal Behaviors

PubMed Central

Du, Jing; Zhu, Ming; Bao, Hongkun; Li, Bai; Dong, Yilong; Xiao, Chunjie; Zhang, Grace Y.; Henter, Ioline; Rudorfer, Matthew; Vitiello, Benedetto

2015-01-01

Numerous studies have linked severe stress to the development of major depressive disorder (MDD), and suicidal behaviors. Furthermore, recent preclinical studies from our laboratory and others have demonstrated that in rodents, chronic stress and the stress hormone cortisol has caused oxidative damage to mitochondrial function and membrane lipids in the brain. Mitochondria play a key role in synaptic neurotransmitter signaling by providing adenosine triphosphate (ATP), mediating lipid and protein synthesis, buffering intracellular calcium, and regulating apoptotic and resilience pathways. Membrane lipids are similarly essential to central nervous system (CNS) function, because cholesterol, polyunsaturated fatty acids, and sphingolipids form a lipid raft region, a special lipid region on the membrane that mediates neurotransmitter signaling through G-protein coupled receptors and ion channels. Low serum cholesterol levels, low antioxidant capacity, and abnormal early morning cortisol levels are biomarkers consistently associated with both depression and suicidal behaviors. In this review, we summarize the manner in which nutrients can protect against oxidative damage to mitochondria and lipids in the neuronal circuits associated with cognitive and affective behaviors. These nutrients include ω3 fatty acids, antioxidants (vitamin C and zinc), members of the vitamin B family (Vitamin B12 and folic acid) and magnesium. Accumulating data have shown that these nutrients can enhance neurocognitive function, and may have therapeutic benefits for depression and suicidal behaviors. A growing body of studies suggests the intriguing possibility that regular consumption of these nutrients may help prevent the onset of mood disorders and suicidal behaviors in vulnerable individuals, or significantly augment the therapeutic effect of available antidepressants. These findings have important implications for the health of both military and civilian populations. PMID:25365455

Energetic costs of protein synthesis do not differ between red- and white-blooded Antarctic notothenioid fishes.

PubMed

Lewis, Johanne M; Grove, Theresa J; O'Brien, Kristin M

2015-09-01

Antarctic icefishes (Family Channichthyidae) within the suborder Notothenioidei lack the oxygen-binding protein hemoglobin (Hb), and six of the 16 species of icefishes lack myoglobin (Mb) in heart ventricle. As iron-centered proteins, Hb and Mb can promote the formation of reactive oxygen species (ROS) that damage biological macromolecules. Consistent with this, our previous studies have shown that icefishes have lower levels of oxidized proteins and lipids in oxidative muscle compared to red-blooded notothenioids. Because oxidized proteins are usually degraded by the 20S proteasome and must be resynthesized, we hypothesized that rates of protein synthesis would be lower in icefishes compared to red-blooded notothenioids, thereby reducing the energetic costs of protein synthesis and conferring a benefit to the loss of Hb and Mb. Rates of protein synthesis were quantified in hearts, and the fraction of oxygen consumption devoted to protein synthesis was measured in isolated hepatocytes and cardiomyocytes of notothenioids differing in the expression of Hb and cardiac Mb. Neither rates of protein synthesis nor the energetic costs of protein synthesis differed among species, suggesting that red-blooded species do not degrade and replace oxidatively modified proteins at a higher rate compared to icefishes but rather, persist with higher levels of oxidized proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Tangeretin exerts anti-neuroinflammatory effects via NF-κB modulation in lipopolysaccharide-stimulated microglial cells.

PubMed

Shu, Zunpeng; Yang, Bingyou; Zhao, Hong; Xu, Bingqing; Jiao, Wenjuan; Wang, Qiuhong; Wang, Zhibin; Kuang, Haixue

2014-04-01

Increasing evidence suggests that tangeretin, a flavonoid from citrus fruit peels, exhibits anti-inflammatory properties and neuroprotective effects in animal disease models. However, the underlying molecular mechanisms are not clearly understood. In this study, we investigated whether tangeretin suppresses excessive microglial activation implicated in the resulting neurotoxicity following stimulation with lipopolysaccharide (LPS) in primary rat microglia and BV-2 microglial cell culture models. The results showed that tangeretin decreased the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), in a dose-dependent manner. Additionally, it inhibited the LPS-induced expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (examined at the protein level) as well as TNF-α, IL-1β, and IL-6 (examined at the mRNA level) in microglial cells. To explore the possible mechanisms underlying these inhibitions by tangeretin, we examined the mitogen-activated protein kinase (MAPK) protein levels and the NF-κB protein signaling pathway. Tangeretin clearly inhibited LPS-induced phosphorylation of ERK, N-terminal Kinase (JNK), and p38. In addition, tangeretin markedly reduced LPS-stimulated phosphorylation of IκB-α and IKK-β, as well as the nuclear translocation of the p65 subunit of pro-inflammatory transcription factor NF-κB. Taken together, these results support further exploration of the therapeutic potential and molecular mechanism of tangeretin in relation to neuroinflammation and neurodegenerative diseases accompanied by microglial activation. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells

PubMed Central

Fischer, Manuel; Horn, Sebastian; Belkacemi, Anouar; Kojer, Kerstin; Petrungaro, Carmelina; Habich, Markus; Ali, Muna; Küttner, Victoria; Bien, Melanie; Kauff, Frank; Dengjel, Jörn; Herrmann, Johannes M.; Riemer, Jan

2013-01-01

Oxidation of cysteine residues to disulfides drives import of many proteins into the intermembrane space of mitochondria. Recent studies in yeast unraveled the basic principles of mitochondrial protein oxidation, but the kinetics under physiological conditions is unknown. We developed assays to follow protein oxidation in living mammalian cells, which reveal that import and oxidative folding of proteins are kinetically and functionally coupled and depend on the oxidoreductase Mia40, the sulfhydryl oxidase augmenter of liver regeneration (ALR), and the intracellular glutathione pool. Kinetics of substrate oxidation depends on the amount of Mia40 and requires tightly balanced amounts of ALR. Mia40-dependent import of Cox19 in human cells depends on the inner membrane potential. Our observations reveal considerable differences in the velocities of mitochondrial import pathways: whereas preproteins with bipartite targeting sequences are imported within seconds, substrates of Mia40 remain in the cytosol for several minutes and apparently escape premature degradation and oxidation. PMID:23676665

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins

PubMed Central

Yao, Chunxiang; Behring, Jessica B.; Shao, Di; Sverdlov, Aaron L.; Whelan, Stephen A.; Elezaby, Aly; Yin, Xiaoyan; Siwik, Deborah A.; Seta, Francesca; Costello, Catherine E.; Cohen, Richard A.; Matsui, Reiko; Colucci, Wilson S.; McComb, Mark E.; Bachschmid, Markus M.

2015-01-01

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a ‘Tandem Mass Tag’ (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation. PMID:26642319

Nanoparticle-induced oxidation of corona proteins initiates an oxidative stress response in cells†

PubMed Central

Jayaram, Dhanya T.; Runa, Sabiha; Kemp, Melissa L.

2017-01-01

Titanium dioxide nanoparticles (TiO2 NPs), used as pigments and photocatalysts, are ubiquitous in our daily lives. Previous work has observed cellular oxidative stress in response to the UV-excitation of photocatalytic TiO2 NPs. In comparison, most human exposure to TiO2 NPs takes place in the dark, in the lung following inhalation or in the gut following consumption of TiO2 NP food pigment. Our spectroscopic characterization shows that both photocatalytic and food grade TiO2 NPs, in the dark, generate low levels of reactive oxygen species (ROS), specifically hydroxyl radicals and superoxides. These ROS oxidize serum proteins that form a corona of proteins on the NP surface. This protein layer is the interface between the NP and the cell. An oxidized protein corona triggers an oxidative stress response, detected with PCR and western blotting. Surface modification of TiO2 NPs to increase or decrease surface defects correlates with ROS generation and oxidative stress, suggesting that NP surface defects, likely oxygen vacancies, are the underlying cause of TiO2 NP-induced oxidative stress. PMID:28537609

Time-resolved method to distinguish protein/peptide oxidation during electrospray ionization mass spectrometry.

PubMed

Pei, Jiying; Hsu, Cheng-Chih; Yu, Kefu; Wang, Yinghui; Huang, Guangming

2018-06-29

Electrospray ionization mass spectrometry (ESI-MS) is one of the most prevalent techniques used to monitor protein/peptide oxidation induced by reactive oxygen species (ROSs). However, both corona discharge (CD) and electrochemistry (EC) can also lead to protein/peptide oxidation during ESI. Because the two types of oxidation occur almost simultaneously, determining the extent to which the two pathways contribute to protein/peptide oxidation is difficult. Herein, a time-resolved method was introduced to identify and differentiate CD- and EC-induced oxidation. Using this approach, we separated the instantaneous CD-induced oxidation from the hysteretic EC-induced oxidation, and the effects of the spray voltage and flow rate of the ESI source on both oxidation types were investigated with a homemade ESI source. For angiotensin II analogue (b-DRVYVHPF-y), the dehydrogenation and oxygenation species were the detected EC-induced oxidation products, while the oxygenation species were the major CD-induced oxidation products. This time-resolved approach was also applicable to a commercial HESI source, in which both CD and EC were responsible for hemoglobin and cytochrome c oxidation with upstream grounding while CD dominated the oxidation without upstream grounding. Copyright © 2018 Elsevier B.V. All rights reserved.

17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha.

PubMed

Batnasan, Enkhzaya; Wang, Ruoxi; Wen, Jitao; Ke, Yueshuang; Li, Xiaoxue; Bohio, Ameer Ali; Zeng, Xianlu; Huo, Hongliang; Han, Liping; Boldogh, Istvan; Ba, Xueqing

2015-01-05

Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H2O2). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H2O2-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Proteomic evaluation of myofibrillar carbonylation in chilled fish mince and its inhibition by catechin.

PubMed

Pazos, Manuel; Maestre, Rodrigo; Gallardo, José M; Medina, Isabel

2013-01-01

The present study investigates the susceptibility of individual myofibrillar proteins from mackerel (Scomber scombrus) mince to undergo carbonylation reactions during chilled storage, and the antioxidant capacity of (+)-catechin to prevent oxidative processes of proteins. The carbonylation of each particular protein was quantified by combining the labelling of protein carbonyls by fluorescein-5-thiosemicarbazide (FTSC) with 1-D or 2-D gel electrophoresis. Alpha skeletal actin, glycogen phosphorylase, unnamed protein product (UNP) similar to enolase, pyruvate kinase, isoforms of creatine kinase, aldolase A and an isoform of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) showed elevated oxidation in chilled non-supplemented mince. Myosin heavy chain (MHC) was not carbonylated in chilled muscle, but an extensive MHC degradation was observed in those samples. The supplementation of catechin reduced protein oxidation and lipid oxidation in a concentration-dependent manner: control>25>100≈200ppm. Therefore, the highest catechin concentrations (100 and 200ppm) exhibited the strongest antioxidant activity. Catechin (200ppm) reduced significantly carbonylation of protein spots identified as glycogen phosphorylase, pyruvate kinase muscle isozyme, isoforms of creatine kinase. Conversely, catechin was ineffective to inhibit the oxidation of actin and UNP similar to enolase. These results draw attention to the inefficiency of catechin to prevent actin oxidation, in contrast to the extremely high efficiency of catechin in inhibiting oxidation of lipids and other proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

A high protein diet upregulated whole-body protein turnover during energy deficit

USDA-ARS?s Scientific Manuscript database

The effects of higher protein diets and sustained energy deficit (ED) on whole-body protein turnover (WBPTO) are not well described. This study examined whether dietary protein level influences whole-body protein breakdown (Ra), non-oxidative leucine disposal (NOLD), and oxidation (Ox) during ED. ...

A Novel Drug Delivery Vesicle Development to Reverse Neurodegeneration: Analysis of the Interactions among Protein, Graphene Oxide and Liposome

NASA Astrophysics Data System (ADS)

Miraz, Md Alamin

In this study, Liposome was decorated with graphene oxide (GO) to synthesize fully-biocompatible theranostic vesicle that can carry bovine serum albumin (BSA) as a model protein. Graphene oxide has been studied as one of the most promising platforms for promoting the growth and repair of neurons. Our graphene oxide based structure could account for the high efficiency of protein loading and deliver to the damaged neuron cell which can reverse the neurodegeneration associated with Alzheimer's disease. The resultant vesicle exhibited high stability in aqueous solution. We investigated the protein adsorption capacity and protein interaction to carbon-based nanomaterials. The Liposome, graphene oxide and bovine serum albumin (BSA) are all biocompatible and hence will not trigger an immune response in vivo.

Strain-dependent effects of long-term treatment with melatonin on kainic acid-induced status epilepticus, oxidative stress and the expression of heat shock proteins.

PubMed

Atanasova, Milena; Petkova, Zlatina; Pechlivanova, Daniela; Dragomirova, Petya; Blazhev, Alexander; Tchekalarova, Jana

2013-10-01

Oxidative stress is implicated in the pathogenesis of both hypertension and epileptogenesis, therefore it could be used as a tool for studying co-morbidity of hypertension and epilepsy. Clinical data suggest that melatonin is a potent antioxidant that is effective in the adjunctive therapy of hypertension and neurodegenerative diseases. The present study aimed to explore and compare the efficacy of chronic pretreatment with melatonin infused via subcutaneous osmotic mini-pumps for 14 days (10 mg/kg per day) on kainic acid (KA)-induced status epilepticus, oxidative stress and expression of heat shock protein (HSP) 72 in spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. SHRs showed higher lipid peroxidation (LP) in the frontal cortex and hippocampus and decreased cytosolic superoxide dismutase (SOD/CuZn) production in the frontal cortex compared to Wistar rats. Status epilepticus (SE) induced by KA (12 mg/kg, i.p.) was accompanied by increased LP and expression of HSP 72 in the hippocampus of the two strains and increased SOD/CuZn production in the frontal cortex of SHRs. Melatonin failed to suppress seizure incidence and intensity though the latency for seizure onset was significantly increased in SHRs. Melatonin attenuated the KA-induced increase in the level of LP in the hippocampus both in SHRs and Wistar rats. However, an increased activity in SOD/CuZn and mitochondrial SOD Mn as well as reduced expression of HSP 72 in the hippocampus was observed only in Wistar rats pretreated with melatonin. Taken together, the observed strain differences in the efficacy of chronic melatonin exposure before SE suggest a lack of a direct link between the seizure activity and the markers of oxidative stress and neurotoxicity. © 2013.

Redox imbalance and mitochondrial abnormalities in the diabetic lung.

PubMed

Wu, Jinzi; Jin, Zhen; Yan, Liang-Jun

2017-04-01

Although the lung is one of the least studied organs in diabetes, increasing evidence indicates that it is an inevitable target of diabetic complications. Nevertheless, the underlying biochemical mechanisms of lung injury in diabetes remain largely unexplored. Given that redox imbalance, oxidative stress, and mitochondrial dysfunction have been implicated in diabetic tissue injury, we set out to investigate mechanisms of lung injury in diabetes. The objective of this study was to evaluate NADH/NAD + redox status, oxidative stress, and mitochondrial abnormalities in the diabetic lung. Using STZ induced diabetes in rat as a model, we measured redox-imbalance related parameters including aldose reductase activity, level of poly ADP ribose polymerase (PAPR-1), NAD + content, NADPH content, reduced form of glutathione (GSH), and glucose 6-phophate dehydrogenase (G6PD) activity. For assessment of mitochondrial abnormalities in the diabetic lung, we measured the activities of mitochondrial electron transport chain complexes I to IV and complex V as well as dihydrolipoamide dehydrogenase (DLDH) content and activity. We also measured the protein content of NAD + dependent enzymes such as sirtuin3 (sirt3) and NAD(P)H: quinone oxidoreductase 1 (NQO1). Our results demonstrate that NADH/NAD + redox imbalance occurs in the diabetic lung. This redox imbalance upregulates the activities of complexes I to IV, but not complex V; and this upregulation is likely the source of increased mitochondrial ROS production, oxidative stress, and cell death in the diabetic lung. These results, together with the findings that the protein contents of DLDH, sirt3, and NQO1 all are decreased in the diabetic lung, demonstrate that redox imbalance, mitochondrial abnormality, and oxidative stress contribute to lung injury in diabetes. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Sigma receptor 1 modulates endoplasmic reticulum stress in retinal neurons.

PubMed

Ha, Yonju; Dun, Ying; Thangaraju, Muthusamy; Duplantier, Jennifer; Dong, Zheng; Liu, Kebin; Ganapathy, Vadivel; Smith, Sylvia B

2011-01-01

To investigate the mechanism of σ receptor 1 (σR1) neuroprotection in retinal neurons. Oxidative stress, which is implicated in diabetic retinopathy, was induced in mouse primary ganglion cells (GCs) and RGC-5 cells, and the effect of the σR1 ligand (+)-pentazocine on pro- and anti-apoptotic and endoplasmic reticulum (ER) stress gene expression was examined. Binding of σR1 to BiP, an ER chaperone protein, and σR1 phosphorylation status were examined by immunoprecipitation. Retinas were harvested from Ins2Akita/+ diabetic mice treated with (+)-pentazocine, and the expression of ER stress genes and of the retinal transcriptome was evaluated. Oxidative stress induced the death of primary GCs and RGC-5 cells. The effect was decreased by the application of (+)-pentazocine. Stress increased σR1 binding to BiP and enhanced σR1 phosphorylation in RGC-5 cells. BiP binding was prevented, and σR1 phosphorylation decreased in the presence of (+)-pentazocine. The ER stress proteins PERK, ATF4, ATF6, IRE1α, and CHOP were upregulated in RGC-5 cells during oxidative stress, but decreased in the presence of (+)-pentazocine. A similar phenomenon was observed in retinas of Ins2Akita/+ diabetic mice. Retinal transcriptome analysis of Ins2Akita/+ mice compared with wild-type revealed differential expression of the genes critically involved in oxidative stress, differentiation, and cell death. The expression profile of those genes was reversed when the Ins2Akita/+ mice were treated with (+)-pentazocine. In retinal neurons, the molecular chaperone σR1 binds BiP under stressful conditions; (+)-pentazocine may exert its effects by dissociating σR1 from BiP. As stress in retinal cells increases, phosphorylation of σR1 is increased, which is attenuated when agonists bind to the receptor.

Copper increases the ability of 6-hydroxydopamine to generate oxidative stress and the ability of ascorbate and glutathione to potentiate this effect: potential implications in Parkinson's disease.

PubMed

Cruces-Sande, Antón; Méndez-Álvarez, Estefanía; Soto-Otero, Ramón

2017-06-01

Copper is an essential metal for the function of many proteins related to important cellular reactions and also involved in the synaptic transmission. Although there are several mechanisms involved in copper homeostasis, a dysregulation in this process can result in serious neurological consequences, including degeneration of dopaminergic neurons. 6-Hydroxydopamine is a dopaminergic neurotoxin mainly used in experimental models of Parkinson's disease, whose neurotoxicity has been related to its ability to generate free radicals. In this study, we examined the effects induced by copper on 6-OHDA autoxidation. Our data show that both Cu + and Cu 2+ caused an increase in • OH production by 6-OHDA autoxidation, which was accompanied by an increase in the rate of both p-quinone formation and H 2 O 2 accumulation. The presence of ascorbate greatly enhanced this process by establishing a redox cycle which regenerates 6-OHDA from its p-quinone. However, the presence of glutathione did not change significantly the copper-induced effects. We observed that copper is able to potentiate the ability of 6-OHDA to cause both lipid peroxidation and protein oxidation, with the latter including a reduction in free-thiol content and an increase in carbonyl content. Ascorbate also increases the lipid peroxidation induced by the action of copper and 6-OHDA. Glutathione protects against the copper-induced lipid peroxidation, but does not reduce its potential to oxidize free thiols. These results clearly demonstrate the potential of copper to increase the capacity of 6-OHDA to generate oxidative stress and the ability of ascorbate to enhance this potential, which may contribute to the destruction of dopaminergic neurons. © 2017 International Society for Neurochemistry.

Roux-en-Y gastric bypass improves glucose homeostasis, reduces oxidative stress and inflammation in livers of obese rats and in Kupffer cells via an AMPK-dependent pathway.

PubMed

Peng, Yanhua; Li, James Zongyu; You, Min; Murr, Michel M

2017-07-01

Oxidative stress and inflammation are implicated in the pathogenesis of steatohepatitis. We hypothesize that Roux-en-Y gastric bypass reduces oxidative stress and inflammation in the liver of obese rats via activation of AMPK-α. Obese Sprague-Dawley male rats underwent either sham operation or Roux-en-Y gastric bypass. Hepatic TNF-α, NF-κB, IRS-2, PI3 kinase, PKC-ζ, NOX2, and AMPK-α were measured. Mechanistic studies were done in a rat Kupffer cell line (RKC1) that was treated with free fatty acids to mimic lipotoxicity and then transfected with AMPK-α siRNA. Reactive oxygen species, TNF-α, NF-κB, AMPK-α, p-AMPK-α, PPAR-γ, and NOX2 were measured. A t test was used. Roux-en-Y gastric bypass lowered nonfasting serum glucose, improved the glucose tolerance test, and induced IRS2/PI3 kinase interaction. Additionally, Roux-en-Y gastric bypass decreased hepatic NOX2, PKC-ζ, TNF-α expression and activation of NF-κB. Free fatty acids increased reactive oxygen species, TNF-α protein, NOX2 protein, and activated NF-κB. Rosiglitazone attenuated the free fatty acids-induced increase in reactive oxygen species, TNF-α, NOX2, and NF-κB; blocking AMPK-α by siRNA abolished the effects of rosiglitazone. Roux-en-Y gastric bypass exhibits antidiabetic properties and is associated with downregulation of proinflammation genes and oxidative stress in the liver and within Kupffer cells via activation of AMPK-α. Copyright © 2017 Elsevier Inc. All rights reserved.

Different oxidative status and expression of calcium channel components in stress-induced dysfunctional chicken muscle.

PubMed

Xing, T; Zhao, X; Wang, P; Chen, H; Xu, X; Zhou, G

2017-04-01

The objective of this study was to assess the effects of transport stress at high ambient temperatures on the oxidation status and the expression of essential elements responsible for the Ca transport (sarco- (endo-) plasmic reticulum Ca-ATPase (SERCA1) and the ryanodine receptor (RyR) in (PM) muscles of broilers. Briefly, Arbor Acres broiler chickens ( = 112) were randomly categorized into 2 treatments: unstressed control (C) and 0.5 h transport (T). Each treatment consisted of 8 replicates of 7 birds each. Birds were transported according to a designed protocol. PM muscle samples in T group were collected and classified as normal (T-NOR) or pale, soft, and exudative-like (T-PSE) using meat quality parameters. The results indicated that production of corticosterone (CORT) and reactive oxygen species (ROS) increased significantly after transportation ( < 0.05). Thiobarbituric acid reactive substance values and carbonyl contents increased significantly in the T group ( < 0.05). Moreover, the extent of lipid peroxidation and protein oxidation was more severe in the T-PSE group compared to the T-NOR group ( < 0.05). The mRNA and protein expression of SERCA1 and αRyR increased in the T-NOR group but decreased significantly in the T-PSE group compared to the CON group ( < 0.05). The mRNA expression of βRyR was found to be enhanced in the T-NOR group compared to the CON group, whereas there was no difference in the T-PSE group ( < 0.05). The results indicate that short-distance transport of broilers affects their physiological responses and biochemical changes which may lead to different oxidative states and, importantly, to different expressions of SERCA and RyR. These induced changes in abnormal sarcoplasmic Ca homeostasis have significant implications for the development of PSE-like meat.

Protein oxidation and peroxidation

PubMed Central

Davies, Michael J.

2016-01-01

Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.

PubMed

Terrill, Jessica R; Radley-Crabb, Hannah G; Iwasaki, Tomohito; Lemckert, Frances A; Arthur, Peter G; Grounds, Miranda D

2013-09-01

The muscular dystrophies comprise more than 30 clinical disorders that are characterized by progressive skeletal muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for pathogenesis generally remains unknown. It is considered that disturbed levels of reactive oxygen species (ROS) contribute to the pathology of many muscular dystrophies. Reactive oxygen species and oxidative stress may cause cellular damage by directly and irreversibly damaging macromolecules such as proteins, membrane lipids and DNA; another major cellular consequence of reactive oxygen species is the reversible modification of protein thiol side chains that may affect many aspects of molecular function. Irreversible oxidative damage of protein and lipids has been widely studied in Duchenne muscular dystrophy, and we have recently identified increased protein thiol oxidation in dystrophic muscles of the mdx mouse model for Duchenne muscular dystrophy. This review evaluates the role of elevated oxidative stress in Duchenne muscular dystrophy and other forms of muscular dystrophies, and presents new data that show significantly increased protein thiol oxidation and high levels of lipofuscin (a measure of cumulative oxidative damage) in dysferlin-deficient muscles of A/J mice at various ages. The significance of this elevated oxidative stress and high levels of reversible thiol oxidation, but minimal myofibre necrosis, is discussed in the context of the disease mechanism for dysferlinopathies, and compared with the situation for dystrophin-deficient mdx mice. © 2013 The Authors Journal compilation © 2013 FEBS.

Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A.

2012-08-21

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the humanmore » protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.« less

Protein carbonylation, protein aggregation and neuronal cell death in a murine model of multiple sclerosis

NASA Astrophysics Data System (ADS)

Dasgupta, Anushka

Many studies have suggested that oxidative stress plays an important role in the pathophysiology of both multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Yet, the mechanism by which oxidative stress leads to tissue damage in these disorders is unclear. Recent work from our laboratory has revealed that protein carbonylation, a major oxidative modification caused by severe and/or chronic oxidative stress conditions, is elevated in MS and EAE. Furthermore, protein carbonylation has been shown to alter protein structure leading to misfolding/aggregation. These findings prompted me to hypothesize that carbonylated proteins, formed as a consequence of oxidative stress and/or decreased proteasomal activity, promote protein aggregation to mediate neuronal apoptosis in vitro and in EAE. To test this novel hypothesis, I first characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of myelin-oligodendrocyte glycoprotein (MOG)35-55 peptide-induced EAE in C57BL/6 mice [Chapter 2]. The results show that carbonylated proteins accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. I discovered not only that there is a temporal correlation between protein carbonylation and apoptosis but also that carbonyl levels are significantly higher in apoptotic cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are also present during the course of EAE, which seems to be due to reduced autophagy. In chapter 3, I show that when gluthathione levels are reduced to those in EAE spinal cord, both neuron-like PC12 (nPC12) cells and primary neuronal cultures accumulate carbonylated proteins and undergo cell death (both by necrosis and apoptosis). Immunocytochemical and biochemical studies also revealed a temporal/spatial relationship between carbonylation, protein aggregation and cellular apoptosis. Furthermore, the effectiveness of the carbonyl scavenger hydralazine, histidine hydrazide and methoxylamine at preventing cell death identifies protein carbonyls as the toxic species. Experiments using well-characterized apoptosis inhibitors place protein carbonylation downstream of the mitochondrial transition pore opening and upstream of caspase activation. These in vitro studies demonstrate for the first time a causal relationship between carbonylation, protein aggregation and apoptosis of neurons undergoing oxidative damage. This relationship was further strengthened with the experiments carried out in chapter 4, which show that inhibition of protein aggregation with congo red (CR) or 2-hydroxypropyl beta-cyclodextrin (HPCD) significantly reduced neuronal cell death without affecting the levels of oxidized proteins. Interestingly, large, juxta-nuclear aggregates are not formed upon GSH depletion, suggesting that the small protein aggregates are the cytotoxic species. Together, our data suggest that protein carbonylation causes protein aggregation to mediate neuronal apoptosis in vitro and that a similar mechanism might be contributing to neuronal/glial apoptosis in EAE. These studies provide the basis for testing protein carbonylation scavengers and protein aggregation inhibitors for the treatment of inflammatory demyelinating disorders.

Thyroid status modulates glycoxidative and lipoxidative modification of tissue proteins.

PubMed

Pamplona, R; Portero-Otín, M; Ruiz, C; Bellmunt, M J; Requena, J R; Thorpe, S R; Baynes, J W; Romero, M; López-Torres, M; Barja, G

1999-10-01

Steady state protein modification by carbonyl compounds is related to the rate of carbonyl adduct formation and the half-life of the protein. Thyroid hormones are physiologic modulators of both tissue oxidative stress and protein degradation. The levels of the glycation product N(epsilon)-fructoselysine (FL) and those of the oxidation products, N(epsilon)-(carboxymethyl)lysine (CML) and malondialdehyde-lysine (MDA-lys), identified by GC/MS in liver proteins, decreased significantly in hyperthyroid rats, as well as (less acutely) in hypothyroid animals. Immunoblotting of liver proteins for advanced glycation end-products (AGE) is in agreement with the results obtained by GC/MS. Cytosolic proteolytic activity against carboxymethylated foreign proteins measured in vitro was significantly increased in hypo- and hyperthyroidism. Oxidative damage to DNA, estimated as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG), did not show significant differences between groups. The results suggests that the steady state levels of these markers depend on the levels of thyroid hormones, presumably through their combined effects on the rates of protein degradation and oxidative stress, whereas DNA is more protected from oxidative damage.

Effect of protein oxidation on the in vitro digestibility of soy protein isolate.

PubMed

Chen, Nannan; Zhao, Mouming; Sun, Weizheng

2013-12-01

Soy protein isolate (SPI) was modified by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) oxidation pretreatment, and the in vitro digestibility of oxidised SPI was investigated. Results indicated that oxidation induced amino acid modification. The amount of most amino acids decreased, accompanied by decreasing digestive proteolysis susceptibility. Peptide size distribution implied that oxidation generated protein aggregates that could not be degraded by pepsin, but could be digested by pancreatin. Oxidation induced a maximum of 16.6% and 14.6% loss, respectively, for free essential and free total amino acid in the digests of oxidised SPI. Antioxidant activities evaluation of oxygen radical absorbance capacity (ORAC) value and DPPH scavenging activity showed that oxidation deteriorated the antioxidant activities of the digests from oxidised SPI. Copyright © 2013 Elsevier Ltd. All rights reserved.

Redox Proteomics of Protein-bound Methionine Oxidation*

PubMed Central

Ghesquière, Bart; Jonckheere, Veronique; Colaert, Niklaas; Van Durme, Joost; Timmerman, Evy; Goethals, Marc; Schymkowitz, Joost; Rousseau, Frederic; Vandekerckhove, Joël; Gevaert, Kris

2011-01-01

We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins. PMID:21406390

Quantifying protein interface footprinting by hydroxyl radical oxidation and molecular dynamics simulation: application to galectin-1.

PubMed

Charvátová, Olga; Foley, B Lachele; Bern, Marshall W; Sharp, Joshua S; Orlando, Ron; Woods, Robert J

2008-11-01

Biomolecular surface mapping methods offer an important alternative method for characterizing protein-protein and protein-ligand interactions in cases in which it is not possible to determine high-resolution three-dimensional (3D) structures of complexes. Hydroxyl radical footprinting offers a significant advance in footprint resolution compared with traditional chemical derivatization. Here we present results of footprinting performed with hydroxyl radicals generated on the nanosecond time scale by laser-induced photodissociation of hydrogen peroxide. We applied this emerging method to a carbohydrate-binding protein, galectin-1. Since galectin-1 occurs as a homodimer, footprinting was employed to characterize the interface of the monomeric subunits. Efficient analysis of the mass spectrometry data for the oxidized protein was achieved with the recently developed ByOnic (Palo Alto, CA) software that was altered to handle the large number of modifications arising from side-chain oxidation. Quantification of the level of oxidation has been achieved by employing spectral intensities for all of the observed oxidation states on a per-residue basis. The level of accuracy achievable from spectral intensities was determined by examination of mixtures of synthetic peptides related to those present after oxidation and tryptic digestion of galectin-1. A direct relationship between side-chain solvent accessibility and level of oxidation emerged, which enabled the prediction of the level of oxidation given the 3D structure of the protein. The precision of this relationship was enhanced through the use of average solvent accessibilities computed from 10 ns molecular dynamics simulations of the protein.

Tryptophan depletion and formation of alpha-aminoadipic and gamma-glutamic semialdehydes in porcine burger patties with added phenolic-rich fruit extracts.

PubMed

Ganhão, Rui; Morcuende, David; Estévez, Mario

2010-03-24

The effect of added fruit extracts on the oxidation of muscle proteins in porcine burger patties subjected to cooking and chill storage was studied. Extracts from arbutus berries (Arbutus unedo L., AU), common hawthorns (Crataegus monogyna L., CM), dog roses (Rosa canina L., RC), and elm-leaf blackberries (Rubus ulmifolius Schott, RU) were prepared, characterized, added to burger patties (3% of total weight), and evaluated as inhibitors of protein oxidation. Negative (no added extract, C) and positive control (added quercetin, 230 mg/kg, Q) groups were also included in the design. Protein oxidation was assessed by means of tryptophan loss using fluorescence spectroscopy (FS) and formation of the specific protein carbonyls alpha-aminoadipic (AAS) and gamma-glutamic semialdehyde (GGS) using liquid chromatography and mass spectroscopy (LC-MS). Both advanced methodologies (FS and LC-MS) were found to be reliable and specific protein oxidation measurements that allow us to gain chemical insight into protein oxidation. The mechanisms likely involved in the oxidative reactions affecting proteins during cooking and storage of burger patties are profusely discussed. Phenolic-rich fruit extracts protected tryptophan residues against oxidation and inhibited the formation of both semialdehydes in burger patties during cooking and subsequent chill storage. In general, RC, RU, and AU were the most effective inhibitors of protein oxidation, with this effect being more intense than that of pure polyphenols like quercetin. These fruit extracts could be considered functional ingredients as their antioxidant actions contribute to the enhancement of the nutritional value of the meat products.

Oxidative protein folding: from thiol-disulfide exchange reactions to the redox poise of the endoplasmic reticulum.

PubMed

Hudson, Devin A; Gannon, Shawn A; Thorpe, Colin

2015-03-01

This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI(red):PDI(ox). The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC-MS-MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment. Copyright © 2014 Elsevier Inc. All rights reserved.

The ubiquitin ligase Nedd4 mediates oxidized low-density lipoprotein-induced downregulation of insulin-like growth factor-1 receptor

PubMed Central

Higashi, Yusuke; Sukhanov, Sergiy; Parthasarathy, Sampath; Delafontaine, Patrice

2008-01-01

Oxidized low-density lipoprotein (LDL) is proatherogenic and induces smooth muscle cell apoptosis, which contributes to atherosclerotic plaque destabilization. We showed previously that oxidized LDL downregulates insulin-like growth factor-1 receptor in human smooth muscle cells and that this is critical for induction of apoptosis. To identify mechanisms, we exposed smooth muscle cells to 60 μg/ml oxidized LDL or native LDL and assessed insulin-like growth factor-1 receptor mRNA levels, protein synthesis rate, and receptor protein stability. Oxidized LDL decreased insulin-like growth factor-1 receptor mRNA levels by 30% at 8 h compared with native LDL, and this decrease was maintained for up to 20 h. However, insulin-like growth factor-1 receptor protein synthesis rate was not altered by oxidized LDL. Pulse-chase labeling experiments revealed that oxidized LDL reduced insulin-like growth factor-1 receptor protein half-life to 12.2 ± 1.7 h from 24.4 ± 4.7 h with native LDL. This destabilization of insulin-like growth factor-1 receptor protein was accompanied by enhanced receptor ubiquitination. Overexpression of dominant-negative Nedd4 prevented oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor, suggesting that Nedd4 was the ubiquitin ligase that mediated receptor downregulation. However, the proteasome inhibitors lactacystin, MG-132, and proteasome inhibitor-1 failed to block oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor. Thus oxidized LDL downregulates insulin-like growth factor-1 receptor by destabilizing the protein via Nedd4-enhanced ubiquitination, leading to degradation via a proteasome-independent pathway. This finding provides novel insights into oxidized LDL-triggered oxidant signaling and mechanisms of smooth muscle cell depletion that contribute to plaque destabilization and coronary events. PMID:18723765

Protein Sulfenylation: A Novel Readout of Environmental Oxidant Stress

PubMed Central

Wages, Phillip A.; Lavrich, Katelyn S.; Zhang, Zhenfa; Cheng, Wan-Yun; Corteselli, Elizabeth; Gold, Avram; Bromberg, Philip; Simmons, Steven O.; Samet, James M.

2016-01-01

Oxidative stress is a commonly cited mechanism of toxicity of environmental agents. Ubiquitous environmental chemicals such as the diesel exhaust component 1,2-naphthoquinone (1,2-NQ) induce oxidative stress by redox cycling, which generates hydrogen peroxide (H2O2). Cysteinyl thiolate residues on regulatory proteins are subjected to oxidative modification by H2O2 in physiological contexts and are also toxicological targets of oxidant stress induced by environmental contaminants. We investigated whether exposure to environmentally relevant concentrations of 1,2-NQ can induce H2O2-dependent oxidation of cysteinyl thiols in regulatory proteins as a readout of oxidant stress in human airway epithelial cells. BEAS-2B cells were exposed to 0–1000 μM 1,2-NQ for 0–30 min, and levels of H2O2 were measured by ratiometric spectrofluorometry of HyPer. H2O2-dependent protein sulfenylation was measured using immunohistochemistry, immunoblotting, and isotopic mass spectrometry. Catalase overexpression was used to investigate the relationship between H2O2 generation and protein sulfenylation in cells exposed to 1,2-NQ. Multiple experimental approaches showed that exposure to 1,2-NQ at concentrations as low as 3 μM induces H2O2-dependent protein sulfenylation in BEAS-2B cells. Moreover, the time of onset and duration of 1,2-NQ-induced sulfenylation of the regulatory proteins GAPDH and PTP1B showed significant differences. Oxidative modification of regulatory cysteinyl thiols in human lung cells exposed to relevant concentrations of an ambient air contaminant represents a novel marker of oxidative environmental stress. PMID:26605980

Protein oxidative modifications during electrospray ionization: solution phase electrochemistry or corona discharge-induced radical attack?

PubMed

Boys, Brian L; Kuprowski, Mark C; Noël, James J; Konermann, Lars

2009-05-15

The exposure of solution-phase proteins to reactive oxygen species (ROS) causes oxidative modifications, giving rise to the formation of covalent +16 Da adducts. Electrospray ionization (ESI) mass spectrometry (MS) is the most widely used method for monitoring the extent of these modifications. Unfortunately, protein oxidation can also take place as an experimental artifact during ESI, such that it may be difficult to assess the actual level of oxidation in bulk solution. Previous work has demonstrated that ESI-induced oxidation is highly prevalent when operating at strongly elevated capillary voltage V(0) (e.g., +8 kV) and with oxygen nebulizer gas in the presence of a clearly visible corona discharge. Protein oxidation under these conditions is commonly attributed to OH radicals generated in the plasma of the discharge. On the other hand, charge balancing oxidation reactions are known to take place at the metal/liquid interface of the emitter. Previous studies have not systematically explored whether such electrochemical processes could be responsible for the formation of oxidative +16 Da adducts instead of (or in combination with) plasma-generated ROS. Using hemoglobin as a model system, this work illustrates the occurrence of extensive protein oxidation even under typical operating conditions (e.g., V(0) = 3.5 kV, N(2) nebulizer gas). Surprisingly, measurements of the current flowing in the ESI circuit demonstrate that a weak corona discharge persists for these relatively gentle settings. On the basis of comparative experiments with nebulizer gases of different dielectric strength, it is concluded that ROS generated under discharge conditions are solely responsible for ESI-induced protein oxidation. This result is corroborated through off-line electrolysis experiments designed to mimic the electrochemical processes taking place during ESI. Our findings highlight the necessity of using easily oxidizable internal standards in biophysical or biomedical ESI-MS studies where knowledge of protein oxidation in bulk solution is desired. Strategies for eliminating ESI-induced oxidation artifacts are discussed.

Detection of Cysteine Redox States in Mitochondrial Proteins in Intact Mammalian Cells.

PubMed

Habich, Markus; Riemer, Jan

2017-01-01

Import, folding, and activity regulation of mitochondrial proteins are important for mitochondrial function. Cysteine residues play crucial roles in these processes as their thiol groups can undergo (reversible) oxidation reactions. For example, during import of many intermembrane space (IMS) proteins, cysteine oxidation drives protein folding and translocation over the outer membrane. Mature mitochondrial proteins can undergo changes in the redox state of specific cysteine residues, for example, as part of their enzymatic reaction cycle or as adaptations to changes of the local redox environment which might influence their activity. Here we describe methods to study changes in cysteine residue redox states in intact cells. These approaches allow to monitor oxidation-driven protein import as well as changes of cysteine redox states in mature proteins during oxidative stress or during the reaction cycle of thiol-dependent enzymes like oxidoreductases.

A strategy for the prevention of protein oxidation by drug product in polymer-based syringes.

PubMed

Nakamura, Koji; Abe, Yoshihiko; Kiminami, Hideaki; Yamashita, Arisa; Iwasaki, Kazuhiro; Suzuki, Shigeru; Yoshino, Keisuke; Dierick, William; Constable, Kevin

2015-01-01

Recently, new and advanced ideas have been presented on the value of polymer-based syringes for improved safety, better strength, reduced aggregation, and the prevention of drug degradation. In this report, our findings on drug degradation from protein oxidation will be presented and discussed. Commonly, dissolved oxygen is one of the factors for causing protein degradation. Due to the nature of higher gas permeability in polymer-based syringes, it was thought to be difficult to control the oxygen level during storage. However, this report demonstrates the appropriateness of combining the use of an oxygen absorber within the secondary packaging as a deoxygenated packaging system. In addition, this report suggests that another factor to enhance protein oxidization is related to radicals on the syringe barrel from sterilization by irradiation. We demonstrate that steam sterilization can minimize protein oxidization, as the protein filled in steam sterilized syringe is much more stable. In conclusion, the main oxidation pathway of a protein has been identified as dissolved oxygen and radical generation within a polymer container. Possible solutions are herewith presented for controlling oxidation by means of applying a deoxygenated packaging system as well as utilizing steam sterilization as a method of sterilization for prefillable polymer syringes. There have been many presentations and discussions about the risks associated with glass prefilled syringes. Advanced ideas are being presented on the value of polymer-based syringes for improved safety, better strength, reduced protein aggregation, and the prevention of drug degradation. Drug degradation based on protein oxidation is discussed in this report. Identification of the main factors causing this degradation and possible solutions available by using polymer-based syringes will be presented. The causes of protein oxidation have been identified as dissolved oxygen and radicals generated by the applied method of sterilization. The oxidation reaction created by dissolved oxygen within the drug product can be effectively inhibited by controlling the removal of the oxygen through the use of a deoxygenated packaging system. This packaging system can control the level or complete removal of oxygen from the primary container and the secondary packaging system. Protein oxidation induced by the formation of radicals from sterilization by irradiation is another critical aspect where it was thought that various sterilization methods were acceptable without loosing drug product quality. However, this report is first to demonstrate that gamma sterilized polymer-based syringes accelerated protein oxidation by radical generation; this effect can be prevented by means of steam sterilization. © PDA, Inc. 2015.

Acute promyelocytic leukemia, arsenic, and PML bodies

PubMed Central

Le Bras, Morgane; Lallemand-Breitenbach, Valérie

2012-01-01

Acute promyelocytic leukemia (APL) is driven by a chromosomal translocation whose product, the PML/retinoic acid (RA) receptor α (RARA) fusion protein, affects both nuclear receptor signaling and PML body assembly. Dissection of APL pathogenesis has led to the rediscovery of PML bodies and revealed their role in cell senescence, disease pathogenesis, and responsiveness to treatment. APL is remarkable because of the fortuitous identification of two clinically effective therapies, RA and arsenic, both of which degrade PML/RARA oncoprotein and, together, cure APL. Analysis of arsenic-induced PML or PML/RARA degradation has implicated oxidative stress in the biogenesis of nuclear bodies and SUMO in their degradation. PMID:22778276

The cell biology of disease: Acute promyelocytic leukemia, arsenic, and PML bodies.

PubMed

de Thé, Hugues; Le Bras, Morgane; Lallemand-Breitenbach, Valérie

2012-07-09

Acute promyelocytic leukemia (APL) is driven by a chromosomal translocation whose product, the PML/retinoic acid (RA) receptor α (RARA) fusion protein, affects both nuclear receptor signaling and PML body assembly. Dissection of APL pathogenesis has led to the rediscovery of PML bodies and revealed their role in cell senescence, disease pathogenesis, and responsiveness to treatment. APL is remarkable because of the fortuitous identification of two clinically effective therapies, RA and arsenic, both of which degrade PML/RARA oncoprotein and, together, cure APL. Analysis of arsenic-induced PML or PML/RARA degradation has implicated oxidative stress in the biogenesis of nuclear bodies and SUMO in their degradation.

ROS-dependent signal transduction

PubMed Central

Reczek, Colleen R; Chandel, Navdeep S

2014-01-01

Reactive oxygen species (ROS) are no longer viewed as just a toxic by-product of mitochondrial respiration, but are now appreciated for their role in regulating a myriad of cellular signaling pathways. H2O2, a type of ROS, is a signaling molecule that confers target specificity through thiol oxidation. Although redox-dependent signaling has been implicated in numerous cellular processes, the mechanism by which the ROS signal is transmitted to its target protein in the face of highly reactive and abundant antioxidants is not fully understood. In this review of redox-signaling biology, we discuss the possible mechanisms for H2O2-dependent signal transduction. PMID:25305438

Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism: implications for breast cancer prevention.

PubMed

Sanchez-Alvarez, Rosa; Martinez-Outschoorn, Ubaldo E; Lin, Zhao; Lamb, Rebecca; Hulit, James; Howell, Anthony; Sotgia, Federica; Rubin, Emanuel; Lisanti, Michael P

2013-01-15

Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with "stemness," more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This "two-compartment" metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert "low-risk" breast cancer patients to "high-risk" status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results also show that antioxidants [such as N-acetyl cysteine (NAC)] can effectively reverse or prevent ethanol-induced oxidative stress in cancer-associated fibroblasts, suggesting a novel strategy for cancer prevention. We also show that caveolin-1 and MCT4 protein expression can be effectively used as new biomarkers to monitor oxidative stress induced by ethanol.

FAD oxidizes the ERO1-PDI electron transfer chain: The role of membrane integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papp, Eszter; Nardai, Gabor; Mandl, Jozsef

2005-12-16

The molecular steps of the electron transfer in the endoplasmic reticulum from the secreted proteins during their oxidation are relatively unknown. We present here that flavine adenine dinucleotide (FAD) is a powerful oxidizer of the oxidoreductase system, Ero1 and PDI, besides the proteins of rat liver microsomes and HepG2 hepatoma cells. Inhibition of FAD transport hindered the action of FAD. Microsomal membrane integrity was mandatory for all FAD-related oxidation steps downstream of Ero1. The PDI inhibitor bacitracin could inhibit FAD-mediated oxidation of microsomal proteins and PDI, but did not hinder the FAD-driven oxidation of Ero1. Our data demonstrated that Ero1more » can utilize FAD as an electron acceptor and that FAD-driven protein oxidation goes through the Ero1-PDI pathway and requires the integrity of the endoplasmic reticulum membrane. Our findings prompt further studies to elucidate the membrane-dependent steps of PDI oxidation and the role of FAD in redox folding.« less

Secondary targets of nitrite-derived reactive nitrogen species: nitrosation/nitration pathways, antioxidant defense mechanisms and toxicological implications.

PubMed

d'Ischia, Marco; Napolitano, Alessandra; Manini, Paola; Panzella, Lucia

2011-12-19

Nitrite, the primary metabolite of nitric oxide (NO) and a widely diffused component of human diet, plays distinct and increasingly appreciated roles in human physiology. However, when exposed to acidic environments, typically in the stomach, or under oxidative stress conditions, it may be converted to a range of reactive nitrogen species (RNS) which in turn can target a variety of biomolecules. Typical consequences of toxicological relevance include protein modification, DNA base deamination and the formation of N-nitrosamines, among the most potent mutagenic and carcinogenic compounds for humans. Besides primary biomolecules, nitrite can cause structural modifications to a variety of endogenous and exogenous organic compounds, ranging from polyunsaturated fatty acids to estrogens, tocopherol, catecholamines, furans, retinoids, dietary phenols, and a range of xenobiotics. The study of the interactions between nitrite and key food components, including phenolic antioxidants, has therefore emerged as an area of great promise for delineating innovative strategies in cancer chemoprevention. Depending on substrates and conditions, diverse reaction pathways may compete to determine product features and distribution patterns. These include nitrosation and nitration but also oxidation, via electron transfer to nitrosonium ion or nitrogen dioxide. This contribution aims to provide an overview of the main classes of compounds that can be targeted by nitrite and to discuss at chemical levels the possible reaction mechanisms under conditions that model those occurring in the stomach. The toxicological implications of the nitrite-modified molecules are finally addressed, and a rational chemical approach to the design of potent antinitrosing agents is illustrated. © 2011 American Chemical Society

Oxidative Stress Induces Disruption of the Axon Initial Segment

PubMed Central

Clark, Kareem C.; Sword, Brooke A.; Dupree, Jeffrey L.

2017-01-01

The axon initial segment (AIS), the domain responsible for action potential initiation and maintenance of neuronal polarity, is targeted for disruption in a variety of central nervous system pathological insults. Previous work in our laboratory implicates oxidative stress as a potential mediator of structural AIS alterations in two separate mouse models of central nervous system inflammation, as these effects were attenuated following reactive oxygen species scavenging and NADPH oxidase-2 ablation. While these studies suggest a role for oxidative stress in modulation of the AIS, the direct effects of reactive oxygen and nitrogen species (ROS/RNS) on the stability of this domain remain unclear. Here, we demonstrate that oxidative stress, as induced through treatment with 3-morpholinosydnonimine (SIN-1), a spontaneous ROS/RNS generator, drives a reversible loss of AIS protein clustering in primary cortical neurons in vitro. Pharmacological inhibition of both voltage-dependent and intracellular calcium (Ca2+) channels suggests that this mechanism of AIS disruption involves Ca2+ entry specifically through L-type voltage-dependent Ca2+ channels and its release from IP3-gated intracellular stores. Furthermore, ROS/RNS-induced AIS disruption is dependent upon activation of calpain, a Ca2+-activated protease previously shown to drive AIS modulation. Overall, we demonstrate for the first time that oxidative stress, as induced through exogenously applied ROS/RNS, is capable of driving structural alterations in the AIS complex. PMID:29228786

Ameliorative Effects of Acacia Honey against Sodium Arsenite-Induced Oxidative Stress in Some Viscera of Male Wistar Albino Rats.

PubMed

Aliyu, Muhammad; Ibrahim, Sani; Inuwa, Hajiya M; Sallau, Abdullahi B; Abbas, Olagunju; Aimola, Idowu A; Habila, Nathan; Uche, Ndidi S

2013-01-01

Cancer is a leading cause of death worldwide and its development is frequently associated with oxidative stress-induced by carcinogens such as arsenicals. Most foods are basically health-promoting or disease-preventing and a typical example of such type is honey. This study was undertaken to investigate the ameliorative effects of Acacia honey on sodium arsenite-induced oxidative stress in the heart, lung and kidney tissues of male Wistar rats. Male Wistar albino rats divided into four groups of five rats each were administered distilled water, Acacia honey (20%), sodium arsenite (5 mg/kg body weight), Acacia honey, and sodium arsenite daily for one week. They were sacrificed anesthetically using 60 mg/kg sodium pentothal. The tissues were used for the assessment of glutathione peroxidase, catalase, and superoxide dismutase activities, protein content and lipid peroxidation. Sodium arsenite significantly (P < 0.05) suppressed the glutathione peroxidase, catalase, superoxide dismutase activities with simultaneous induction of lipid peroxidation. Administration of Acacia honey significantly increased (P < 0.05) glutathione peroxidase, catalase, and superoxide dismutase activities with concomitant suppression of lipid peroxidation as evident by the decrease in malondialdehyde level. From the results obtained, Acacia honey mitigates sodium arsenite induced-oxidative stress in male Wistar albino rats, which suggest that it may attenuate oxidative stress implicated in chemical carcinogenesis.

Hydroxyl radical and ferryl-generating systems promote gel network formation of myofibrillar protein.

PubMed

Xiong, Youling L; Blanchard, Suzanne P; Ooizumi, Tooru; Ma, Yuanyuan

2010-03-01

The objective of the study was to examine how oxidatively induced protein cross-linking would influence the gelation properties of myofibrillar protein (MP) under meat processing conditions. MP suspensions in 0.6 M NaCl at pH 6 were treated with an iron-catalyzed oxidizing system (IOS: 10 microM FeCl(3), 0.1 mM ascorbic acid, 0.05 to 5 mM H(2)O(2)) or a H(2)O(2)-activated metmyoglobin oxidizing system (MOS: 0.01 to 0.1 mM metmyoglobin/H(2)O(2)) that produced hydroxyl radical and ferryl species, respectively. Both oxidizing systems promoted MP thermal gelation, which was evidenced by rapid protein-protein interaction and the enhancement in storage modulus (elasticity) of the gel network as revealed by dynamic rheological testing in the 20 to 74 degrees C temperature range. This gelation-enhancing effect was attributed to the shift of myosin aggregation in the early stage of heating from predominantly head-head association (nonoxidized control samples) to prevalently tail-tail cross-linking through disulfide bonds. However, both hardness and water-holding capacity of chilled gels tended to decline when MP was exposed to >or=1 mM H(2)O(2) in IOS and to all concentrations of metmyoglobin in MOS. Microscopic examination confirmed a more porous structure in oxidized gels when compared with nonoxidized protein gels. The results demonstrated that mild oxidation altered the mode of myosin aggregation in favor of an elastic gel network formation, but it did not improve or had a negative effect on water-binding properties of MP gels. Mild oxidation promotes protein network formation and enhances gelation of myofibrillar protein under normal salt and pH conditions used in meat processing. This oxidative effect, which involves disulfide linkages, is somewhat similar to that in bakery product processing where oxidants are used to improve dough performance through gluten protein interaction.

Hypothermia can reverse hepatic oxidative stress damage induced by hypoxia in rats.

PubMed

Garnacho-Castaño, Manuel Vicente; Alva, Norma; Sánchez-Nuño, Sergio; Bardallo, Raquel G; Palomeque, Jesús; Carbonell, Teresa

2016-12-01

Our previous findings demonstrated that hypothermia enhances the reduction potential in the liver and helps to maintain the plasmatic antioxidant pool. Here, we aimed to elucidate if hypothermia protects against hypoxia-induced oxidative stress damage in rat liver. Several hepatic markers of oxidative stress were compared in three groups of animals (n = 8 in each group): control normothermic group ventilated with room air and two groups under extreme hypoxia (breathing 10 % O 2 ), one kept at normothermia (HN) (37 °C) and the other under deep hypothermia (HH) (central body temperature of 21-22 °C). Hypoxia in normothermia significantly increased the levels of hepatic nitric oxide, inducible nitric oxide synthase expression, protein oxidation, Carbonilated proteins, advanced oxidation protein products, 4-hydroxynonenal (HNE) protein adducts, and lipid peroxidation when compared to the control group (p < 0.05). However, when hypoxia was induced under hypothermia, results from the oxidative stress biomarker analyses did not differ significantly from those found in the control group. Indeed, 4-HNE protein adduct amounts were significantly lower in the HH versus HN group (p < 0.05). Therefore, hypothermia can mitigate hypoxia-induced oxidative stress damage in rat liver. These effects could help clarify the mechanisms of action of therapeutic hypothermia.

HIV-1 viral protein R (Vpr) induces fatty liver in mice via LXRα and PPARα dysregulation: implications for HIV-specific pathogenesis of NAFLD.

PubMed

Agarwal, Neeti; Iyer, Dinakar; Gabbi, Chiara; Saha, Pradip; Patel, Sanjeet G; Mo, Qianxing; Chang, Benny; Goswami, Biman; Schubert, Ulrich; Kopp, Jeffrey B; Lewis, Dorothy E; Balasubramanyam, Ashok

2017-10-17

HIV patients develop hepatic steatosis. We investigated hepatic steatosis in transgenic mice expressing the HIV-1 accessory protein Vpr (Vpr-Tg) in liver and adipose tissues, and WT mice infused with synthetic Vpr. Vpr-Tg mice developed increased liver triglyceride content and elevated ALT, bilirubin and alkaline phosphatase due to three hepatic defects: 1.6-fold accelerated de novo lipogenesis (DNL), 45% slower fatty acid ß-oxidation, and 40% decreased VLDL-triglyceride export. Accelerated hepatic DNL was due to coactivation by Vpr of liver X receptor-α (LXRα) with increased expression of its lipogenic targets Srebp1c, Chrebp, Lpk, Dgat, Fasn and Scd1, and intranuclear SREBP1c and ChREBP. Vpr enhanced association of LXRα with Lxrα and Srebp1c promoters, increased LXRE-LXRα binding, and broadly altered hepatic expression of LXRα-regulated lipid metabolic genes. Diminished hepatic fatty acid ß-oxidation was associated with decreased mRNA expression of Pparα and its targets Cpt1, Aox, Lcad, Ehhadh, Hsd10 and Acaa2, and blunted VLDL export with decreased expression of Mttp and its product microsomal triglyceride transfer protein. With our previous findings that Vpr circulates in HIV patients (including those with undetectable plasma HIV-1 RNA), co-regulates the glucocorticoid receptor and PPARγ and transduces hepatocytes, these data indicate a potential role for Vpr in HIV-associated fatty liver disease.

Calcium-sensitive regulation of monoamine oxidase-A contributes to the production of peroxyradicals in hippocampal cultures: implications for Alzheimer disease-related pathology

PubMed Central

Cao, Xia; Wei, Zelan; Gabriel, Geraldine G; Li, XinMin; Mousseau, Darrell D

2007-01-01

Background Calcium (Ca2+) has recently been shown to selectively increase the activity of monoamine oxidase-A (MAO-A), a mitochondria-bound enzyme that generates peroxyradicals as a natural by-product of the deamination of neurotransmitters such as serotonin. It has also been suggested that increased intracellular free Ca2+ levels as well as MAO-A may be contributing to the oxidative stress associated with Alzheimer disease (AD). Results Incubation with Ca2+ selectively increases MAO-A enzymatic activity in protein extracts from mouse hippocampal HT-22 cell cultures. Treatment of HT-22 cultures with the Ca2+ ionophore A23187 also increases MAO-A activity, whereas overexpression of calbindin-D28K (CB-28K), a Ca2+-binding protein in brain that is greatly reduced in AD, decreases MAO-A activity. The effects of A23187 and CB-28K are both independent of any change in MAO-A protein or gene expression. The toxicity (via production of peroxyradicals and/or chromatin condensation) associated with either A23187 or the AD-related β-amyloid peptide, which also increases free intracellular Ca2+, is attenuated by MAO-A inhibition in HT-22 cells as well as in primary hippocampal cultures. Conclusion These data suggest that increases in intracellular Ca2+ availability could contribute to a MAO-A-mediated mechanism with a role in AD-related oxidative stress. PMID:17868476

Late-onset dietary restriction compensates for age-related increase in oxidative stress and alterations of HSP 70 and synapsin 1 protein levels in male Wistar rats.

PubMed

Sharma, Sandeep; Singh, Rumani; Kaur, Manpreet; Kaur, Gurcharan

2010-04-01

Numerous reports implicate increased oxidative stress in the functional and structural changes occurring in the brain and other organs as a part of the normal aging process. Dietary restriction (DR) has long been shown to be life-prolonging intervention in several species. This study was aimed to assess the potential efficacy of late-onset short term DR when initiated in 21 months old male wistar rats for 3 months on the antioxidant defense system and lipid peroxidation, cellular stress response protein HSP 70 and synaptic marker protein synapsin 1 in discrete brain regions such as cortex, hypothalamus, and hippocampus as well as liver, kidney and heart from 24 month old rats. Age-associated decline in activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione, and elevated levels of lipid peroxidation was observed in brain and peripheral organ as well as increased expression of HSP 70 and reduction in synapsin 1 was observed in brain studied. Late-onset short term DR was effective in partially restoring the antioxidant status and in decreasing lipid peroxidation level as well as enhancing the expression of HSP 70 and synapsin 1 in aged rats. Late onset short term DR also prevented age-related neurodegeneration as revealed by Fluoro-Jade B staining in hippocampus and cortex regions of rat brain. Thus our current results suggest that DR initiated even in old age has the potential to improve age related decline in body functions.

Improvement of Tissue Survival of Skin Flaps by 5α-Reductase Inhibitors: Possible Involvement of Nitric Oxide and Inducible Nitric Oxide Synthase

PubMed Central

Karimi, Ali Asghar; Ajami, Marjan; Asadi, Yasin; Aboutaleb, Nahid; Gorjipour, Fazel; Malekloo, Roya; Pazoki-Toroudi, Hamidreza

2015-01-01

Background: Skin flap grafting is a popular approach for reconstruction of critical skin and underlying soft tissue injuries. In a previous study, we demonstrated the beneficial effects of two 5α-reductase inhibitors, azelaic acid and finasteride, on tissue survival in a rat model of skin flap grafting. In the current study, we investigated the involvement of nitric oxide and inducible nitric oxide synthase (iNOS) in graft survival mediated by these agents. Methods: A number of 42 male rats were randomly allocated into six groups: 1, normal saline topical application; 2, azelaic acid (100 mg/flap); 3, finasteride (1 mg/flap); 4, injection of L-NG-nitroarginine methyl ester (L-NAME) (i.p., 20 mg/kg); 5, L-NAME (20 mg/kg, i.p.) + azelaic acid (100 mg/flap, topical); 6, L-NAME (20 mg/kg, i.p.) + finasteride (1 mg/flap, topical). Tissue survival, level of nitric oxide, and iNOS expression in groups were measured. Results: Our data revealed that azelaic acid and finasteride significantly increased the expression of iNOS protein and nitric oxide (NO) levels in graft tissue (P < 0.05). These increases in iNOS expression and NO level were associated with higher survival of the graft tissue. Conclusion: It appears that alterations of the NO metabolism are implicated in the azelaic acid- and finasteride-mediated survival of the skin flaps. PMID:25864816

Application of confocal Raman micro-spectroscopy for label-free monitoring of oxidative stress in living bronchial cells

NASA Astrophysics Data System (ADS)

Surmacki, Jakub M.; Quirós Gonzalez, Isabel; Bohndiek, Sarah E.

2018-02-01

Oxidative stress in cancer is implicated in tumor progression, being associated with increased therapy resistance and metastasis. Conventional approaches for monitoring oxidative stress in tissue such as high-performance liquid chromatography and immunohistochemistry are bulk measurements and destroy the sample, meaning that longitudinal monitoring of cancer cell heterogeneity remains elusive. Raman spectroscopy has the potential to overcome this challenge, providing a chemically specific, label free readout from single living cells. Here, we applied a standardized protocol for label-free confocal Raman micro-spectroscopy in living cells to monitor oxidative stress in bronchial cells. We used a quartz substrate in a commercial cell chamber contained within a microscope incubator providing culture media for cell maintenance. We studied the effect of a potent reactive oxygen species inducer, tert-butyl hydroperoxide (TBHP), and antioxidant, N-acetyl-L-cysteine (NAC) on living cells from a human bronchial epithelial cells (HBEC). We found that the Raman bands corresponding to nucleic acids, proteins and lipids were significantly different (p<0.05) for control, TBHP, and NAC. Encouragingly, partial least squares discriminant analysis applied to our data showed high sensitivity and specificity for identification of control (87.3%, 71.7%), NAC (92.3%, 85.1%) and TBHP (86.9%, 92.9%). These results suggest that confocal Raman micro-spectroscopy may be able to monitor the biological impact of oxidative and reductive processes in cells, hence enabling longitudinal studies of oxidative stress in therapy resistance and metastasis at the single cell level.

Evaluation of three simple direct or indirect carbonyl detection methods for characterization of oxidative modifications of proteins.

PubMed

Vásquez-Garzón, Verónica R; Rouimi, Patrick; Jouanin, Isabelle; Waeg, Georg; Zarkovic, Neven; Villa-Treviño, Saul; Guéraud, Françoise

2012-05-01

Among disruptions induced by oxidative stress, modifications of proteins, particularly irreversible carbonylation, are associated with the development of several diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Carbonylation of proteins can occur directly or indirectly through the adduction of lipid oxidation products. In this study, three classical and easy-to-perform techniques to detect direct or indirect carbonylation of proteins were compared. A model protein apomyoglobin and a complex mixture of rat liver cytosolic proteins were exposed to cumene hydroperoxide oxidation or adduction to the lipid peroxidation product 4-hydroxynonenal in order to test direct or indirect carbonylation, respectively. The technique using a specific anti-4-hydroxynonenal-histidine adduct antibody was effective to detect in vitro modification of model apomyoglobin and cytosolic proteins by 4-hydroxynonenal but not by direct carbonylation which was achieved by techniques using biotin-coupled hydrazide or dinitrophenylhydrazine derivatization of carbonyls. Sequential use of these methods enabled the detection of both direct and indirect carbonyl modification in proteins, although constitutively biotinylated proteins were detected by biotin-hydrazide. Although rather classical and efficient, methods for carbonyl detection on proteins in oxidative stress studies may be biased by some artifactual detections and complicated by proteins multimerizations. The use of more and more specific available antibodies is recommended to complete detection of lipid peroxidation product adducts on proteins.

Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling*

PubMed Central

Jayakumar, Arumugam R.; Liu, Mingli; Moriyama, Mitsuaki; Ramakrishnan, Ramugounder; Forbush, Bliss; Reddy, Pichili V. B.; Norenberg, Michael D.

2008-01-01

Brain edema and the consequent increase in intracranial pressure and brain herniation are major complications of acute liver failure (fulminant hepatic failure) and a major cause of death in this condition. Ammonia has been strongly implicated as an important factor, and astrocyte swelling appears to be primarily responsible for the edema. Ammonia is known to cause cell swelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter (NKCC1) has been shown to be involved in cell swelling in several neurological disorders. We therefore examined the effect of ammonia on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to ammonia (NH4Cl; 5 mm), and NKCC activity was measured. Ammonia increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished ammonia-induced astrocyte swelling. Ammonia also increased total as well as phosphorylated NKCC1. Treatment with cyclohexamide, a potent inhibitor of protein synthesis, diminished NKCC1 protein expression and NKCC activity. Since ammonia is known to induce oxidative/nitrosative stress, and antioxidants and nitric-oxide synthase inhibition diminish astrocyte swelling, we also examined whether ammonia caused oxidation and/or nitration of NKCC1. Cultures exposed to ammonia increased the state of oxidation and nitration of NKCC1, whereas the antioxidants N-nitro-l-arginine methyl ester and uric acid all significantly diminished NKCC activity. These agents also reduced phosphorylated NKCC1 expression. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by ammonia and that such activation appears to be mediated by NKCC1 abundance as well as by its oxidation/nitration and phosphorylation. PMID:18849345

Distinct Growth and Secretome Strategies for Two Taxonomically Divergent Brown Rot Fungi.

PubMed

Presley, Gerald N; Schilling, Jonathan S

2017-04-01

Brown rot fungi are wood-degrading fungi that employ both oxidative and hydrolytic mechanisms to degrade wood. Hydroxyl radicals that facilitate the oxidative component are powerful nonselective oxidants and are incompatible with hydrolytic enzymes unless they are spatially segregated in wood. Differential gene expression has been implicated in the segregation of these reactions in Postia placenta , but it is unclear if this two-step mechanism varies in other brown rot fungi with different traits and life history strategies that occupy different niches in nature. We employed proteomics to analyze a progression of wood decay on thin wafers, using brown rot fungi with significant taxonomic and niche distances: Serpula lacrymans (Boletales; "dry rot" lumber decay) and Gloeophyllum trabeum (order Gloeophyllales; slash, downed wood). Both fungi produced greater oxidoreductase diversity upon wood colonization and greater glycoside hydrolase activity later, consistent with a two-step mechanism. The two fungi invested very differently, however, in terms of growth (infrastructure) versus protein secretion (resource capture), with the ergosterol/extracted protein ratio being 7-fold higher with S. lacrymans than with G. trabeum In line with the native substrate associations of these fungi, hemicellulase-specific activities were dominated by mannanase in S. lacrymans and by xylanase in G. trabeum Consistent with previous observations, S. lacrymans did not produce glycoside hydrolase 6 (GH6) cellobiohydrolases (CBHs) in this study, despite taxonomically belonging to the order Boletales, which is distinguished among brown rot fungi by having CBH genes. This work suggests that distantly related brown rot fungi employ staggered mechanisms to degrade wood, but the underlying strategies vary among taxa. IMPORTANCE Wood-degrading fungi are important in forest nutrient cycling and offer promise in biotechnological applications. Brown rot fungi are unique among these fungi in that they use a nonenzymatic oxidative pretreatment before enzymatic carbohydrate hydrolysis, enabling selective removal of carbohydrates from lignin. This capacity has independently evolved multiple times, but it is unclear if different mechanisms underpin similar outcomes. Here, we grew fungi directionally on wood wafers and we found similar two-step mechanisms in taxonomically divergent brown rot fungi. The results, however, revealed strikingly different growth strategies, with S. lacrymans investing more in biomass production than secretion of proteins and G. trabeum showing the opposite pattern, with a high diversity of uncharacterized proteins. The "simplified" S. lacrymans secretomic system could help narrow gene targets central to oxidative brown rot pretreatments, and a comparison of its distinctions with G. trabeum and other brown rot fungi (e.g., Postia placenta ) might offer similar traction in noncatabolic genes. Copyright © 2017 American Society for Microbiology.

Promyelocytic Leukemia Protein, a Protein at the Crossroad of Oxidative Stress and Metabolism.

PubMed

Tessier, Sarah; Martin-Martin, Natalia; de Thé, Hugues; Carracedo, Arkaitz; Lallemand-Breitenbach, Valérie

2017-03-20

Cellular metabolic activity impacts the production of reactive oxygen species (ROS), both positively through mitochondrial oxidative processes and negatively by promoting the production of reducing agents (including NADPH and reduced glutathione). A defined metabolic state in cancer cells is critical for cell growth and long-term self-renewal, and such state is intrinsically associated with redox balance. Promyelocytic leukemia protein (PML) regulates several biological processes, at least in part, through its ability to control the assembly of PML nuclear bodies (PML NBs). Recent Advances: PML is oxidation-prone, and oxidative stress promotes NB biogenesis. These nuclear subdomains recruit many nuclear proteins and regulate their SUMOylation and other post-translational modifications. Some of these cargos-such as p53, SIRT1, AKT, and mammalian target of rapamycin (mTOR)-are key regulators of cell fate. PML was also recently shown to regulate oxidation. While it was long considered primarily as a tumor suppressor protein, PML-regulated metabolic switch uncovered that this protein could promote survival and/or stemness of some normal or cancer cells. In this study, we review the recent findings on this multifunctional protein. Studying PML scaffolding functions as well as its fine role in the activation of p53 or fatty acid oxidation will bring new insights in how PML could bridge oxidative stress, senescence, cell death, and metabolism. Antioxid. Redox Signal. 26, 432-444.

UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products

PubMed Central

Linetsky, Mikhail; Raghavan, Cibin T.; Johar, Kaid; Fan, Xingjun; Monnier, Vincent M.; Vasavada, Abhay R.; Nagaraj, Ram H.

2014-01-01

Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320–400 nm, 100 milliwatts/cm2, 45 min to 2 h), young human lenses (20–36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans. PMID:24798334

Polycystic ovary syndrome offspring display increased oxidative stress markers comparable to gestational diabetes offspring.

PubMed

Boutzios, Georgios; Livadas, Sarantis; Piperi, Christina; Vitoratos, Nicolaos; Adamopoulos, Christos; Hassiakos, Dimitrios; Iavazzo, Christos; Diamanti-Kandarakis, Evanthia

2013-03-01

To study oxidative stress (OS) markers on neonates. The specific aim was to evaluate advanced glycation end products (AGEs) and advanced oxidation protein products (AOPPs) serum levels along with the hormonal/metabolic profile and their possible relationship in a cohort of polycystic ovary syndrome PCOS(N) and gestational diabetes GDM(N) neonates and their mothers PCOS(M) and GDM(M). Prospective controlled study. Academic medical center. The study population comprised 151 mother/neonate pairs. Diet and/or insulin administration in GDM(M). Anthropometric, metabolic, hormonal parameters, and OS markers. The AGEs and AOPPs were higher in PCOS(M) and GDM(M) compared with controls (M). The same significant difference was observed in the corresponding groups of neonates. A strong relationship between mothers and neonates regarding AGEs (r = 0.605) and AOPPs levels (r = 0.735) was disclosed. Analogous findings were observed regarding androgens and insulin resistance in mothers and neonates, respectively. The present study demonstrated that in PCOS(N), the OS status was similar to that of GDM(N) and strongly associated with their mothers' oxidative status. These findings may have clinical implications, as exposure of PCOS(N) to high OS levels during pregnancy could affect several health issues of neonates. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Fructose-enriched diet induces inflammation and reduces antioxidative defense in visceral adipose tissue of young female rats.

PubMed

Kovačević, Sanja; Nestorov, Jelena; Matić, Gordana; Elaković, Ivana

2017-02-01

The consumption of refined, fructose-enriched food continuously increases and has been linked to development of obesity, especially in young population. Low-grade inflammation and increased oxidative stress have been implicated in the pathogenesis of obesity-related disorders including type 2 diabetes. In this study, we examined alterations in inflammation and antioxidative defense system in the visceral adipose tissue (VAT) of fructose-fed young female rats, and related them to changes in adiposity and insulin sensitivity. We examined the effects of 9-week fructose-enriched diet applied immediately after weaning on nuclear factor κB (NF-κB) intracellular distribution, and on the expression of pro-inflammatory cytokines (IL-1β and TNFα) and key antioxidative enzymes in the VAT of female rats. Insulin signaling in the VAT was evaluated at the level of insulin receptor substrate-1 (IRS-1) protein and its inhibitory phosphorylation on Ser 307 . Fructose-fed rats had increased VAT mass along with increased NF-κB nuclear accumulation and elevated IL-1β, but not TNFα expression. The protein levels of antioxidative defense enzymes, mitochondrial manganese superoxide dismutase 2, and glutathione peroxidase, were reduced, while the protein content of IRS-1 and its inhibitory phosphorylation were not altered by fructose diet. The results suggest that fructose overconsumption-related alterations in pro-inflammatory markers and antioxidative capacity in the VAT of young female rats can be implicated in the development of adiposity, but do not affect inhibitory phosphorylation of IRS-1.

Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.

PubMed

Sato, Yoshimi; Kojima, Rieko; Okumura, Masaki; Hagiwara, Masatoshi; Masui, Shoji; Maegawa, Ken-ichi; Saiki, Masatoshi; Horibe, Tomohisa; Suzuki, Mamoru; Inaba, Kenji

2013-01-01

The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4), and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46, and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4-PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

Effects of quality of energy on substrate oxidation in enterally fed, low-birth-weight infants.

PubMed

Kashyap, S; Towers, H M; Sahni, R; Ohira-Kist, K; Abildskov, K; Schulze, K F

2001-09-01

Carbohydrate and fat may differ in their ability to support energy-requiring physiologic processes, such as protein synthesis and growth. If so, varying the constituents of infant formula might be therapeutically advantageous. We tested the hypothesis that low-birth-weight infants fed a diet containing 65% of nonprotein energy as carbohydrate oxidize relatively more carbohydrate and relatively less protein than do infants fed an isoenergetic, isonitrogenous diet containing 35% of nonprotein energy as carbohydrate. Sixty-two low-birth-weight infants weighing from 750 to 1600 g at birth were assigned randomly and blindly to receive 1 of 5 formulas that differed only in the quantity and quality of nonprotein energy. Formula containing 544 kJ x kg(-1) x d(-1) with either 50%, 35%, or 65% of nonprotein energy as carbohydrate was administered to control subjects, group 1, and group 2, respectively. Groups 3 and 4 received gross energy intakes of 648 kJ x kg(-1) x d(-1) with 35% and 65% of nonprotein energy as carbohydrate. Protein intake was targeted at 4 g x kg(-1) x d(-1). Substrate oxidation was estimated from biweekly, 6-h measurements of gas exchange and 24-h urinary nitrogen excretion. Carbohydrate oxidation was positively (r = 0.71, P < 0.0001) and fat oxidation was negatively (r = -0.46, P < 0.001) correlated with carbohydrate intake. Protein oxidation was negatively correlated with carbohydrate oxidation (r = -0.42, P < 0.001). Fat oxidation was not correlated with protein oxidation. Protein oxidation was less in infants receiving 65% of nonprotein energy as carbohydrate than in groups receiving 35% nonprotein energy as carbohydrate. These data support the hypothesis that energy supplied as carbohydrate is more effective than energy supplied as fat in sparing protein oxidation in enterally fed low-birth-weight infants.

Ceruloplasmin inhibits carbonyl formation in endogenous proteins in phorbol myristate acetate (PMA)-stimulated neutrophils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krsek-Staples, J.; Webster, R.O.

1991-03-11

The respiratory burst of stimulated neutrophils can cause oxidative modifications of endogenous neutrophil proteins as measured by increased carbonyl formation. Ceruloplasmin is an acute phase protein and may act as an antioxidant during inflammation. Therefore, the role of ceruloplasmin in preventing oxidative damage of endogenous neutrophil proteins was investigated. Protein carbonyl content was determined spectrophotometrically using 2,4-dinitrophenylhydrazine. Ceruloplasmin, at a concentration present during inflammation significantly inhibited carbonyl formation in endogenous proteins of PMA-stimulated neutrophils. In order to determine if oxidative damage was occurring to the ceruloplasmin upon incubation with stimulated neutrophils, carbonyl formation in the ceruloplasmin in the presence andmore » absence of stimulated neutrophils. This data suggests that ceruloplasmin may play a role in regulating oxidative damage to proteins and that ceruloplasmin itself may act as a target for these modifications.« less

Reduced protein oxidation in Wistar rats supplemented with marine ω3 PUFAs.

PubMed

Méndez, Lucía; Pazos, Manuel; Gallardo, José M; Torres, Josep L; Pérez-Jiménez, Jara; Nogués, Rosa; Romeu, Marta; Medina, Isabel

2013-02-01

The potential effects of various dietary eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) ratios (1:1, 2:1, and 1:2, respectively) on protein redox states from plasma, kidney, skeletal muscle, and liver were investigated in Wistar rats. Dietary fish oil groups were compared with animals fed soybean and linseed oils, vegetable oils enriched in ω6 linoleic acid (LA; 18:2) and ω3 α-linolenic acid (ALA; 18:3), respectively. Fish oil treatments were effective at reducing the level of total fatty acids in plasma and enriching the plasmatic free fatty acid fraction and erythrocyte membranes in EPA and DHA. A proteomic approach consisting of fluorescein 5-thiosemicarbazide (FTSC) labeling of protein carbonyls, FTSC intensity visualization on 1-DE or 2-DE gels, and protein identification by MS/MS was used for the protein oxidation assessment. Albumin was found to be the most carbonylated protein in plasma for all dietary groups, and its oxidation level was significantly modulated by dietary interventions. Supplementation with an equal EPA:DHA ratio (1:1) showed the lowest oxidation score for plasma albumin, followed in increasing order of carbonylation by 1:2 <2:1 ≈ linseed < soybean. Oxidation patterns of myofibrillar skeletal muscle proteins and cytosolic proteins from kidney and liver also indicated a protective effect on proteins for the fish oil treatments, the 1:1 ratio exhibiting the lowest protein oxidation scores. The effect of fish oil treatments at reducing carbonylation on specific proteins from plasma (albumin), skeletal muscle (actin), and liver (albumin, argininosuccinate synthetase, 3-α-hydroxysteroid dehydrogenase) was remarkable. This investigation highlights the efficiency of dietary fish oil at reducing in vivo oxidative damage of proteins compared to oils enriched in the 18-carbon polyunsaturated fatty acids ω3 ALA and ω6 LA, and such antioxidant activity may differ among different fish oil sources because of variations in EPA/DHA content. Copyright © 2012 Elsevier Inc. All rights reserved.

Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques.

PubMed Central

Woods, Alan A; Linton, Stuart M; Davies, Michael J

2003-01-01

Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has been interpreted in terms of the occurrence of two oxidative mechanisms, one involving oxygen-derived radicals catalysed by trace transition metal ions, and a second involving chlorinating species (HOCl or Cl2), generated by the haem enzyme myeloperoxidase (MPO). As MPO is released extracellularly by activated monocytes (and possibly macrophages) and is a highly basic protein, it would be expected to associate with polyanions such as the glycosaminoglycans of the extracellular matrix, and might result in damage being localized at such sites. In this study proteins extracted from extracellular matrix material obtained from advanced human atherosclerotic lesions are shown to contain elevated levels of oxidized amino acids [3,4-dihydroxyphenylalanine (DOPA), di-tyrosine, 2-hydroxyphenylalanine ( o-Tyr)] when compared with healthy (human and pig) arterial tissue. These matrix-derived materials account for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions. The detection of elevated levels of DOPA and o-Tyr, which have been previously attributed to the occurrence of oxygen-radical-mediated reactions, by HOCl treatment, suggests an alternative route to the formation of these materials in plaques. This is believed to involve the formation and subsequent decomposition of protein chloramines. PMID:12456264

Increased protein oxidation in human substantia nigra pars compacta in comparison with basal ganglia and prefrontal cortex measured with an improved dinitrophenylhydrazine assay.

PubMed

Floor, E; Wetzel, M G

1998-01-01

The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4-dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size-dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.

Nuclear ferritin: A new role for ferritin in cell biology.

PubMed

Alkhateeb, Ahmed A; Connor, James R

2010-08-01

Ferritin has been traditionally considered a cytoplasmic iron storage protein. However, several studies over the last two decades have reported the nuclear localization of ferritin, specifically H-ferritin, in developing neurons, hepatocytes, corneal epithelial cells, and some cancer cells. These observations encouraged a new perspective on ferritin beyond iron storage, such as a role in the regulation of iron accessibility to nuclear components, DNA protection from iron-induced oxidative damage, and transcriptional regulation. This review will address the translocation and functional significance of nuclear ferritin in the context of human development and disease. The nuclear translocation of ferritin is a selective energy-dependent process that does not seem to require a consensus nuclear localization signal. It is still unclear what regulates the nuclear import/export of ferritin. Some reports have implicated the phosphorylation and O-glycosylation of the ferritin protein in nuclear transport; others suggested the existence of a specific nuclear chaperone for ferritin. The data argue strongly for nuclear ferritin as a factor in human development and disease. Ferritin can bind and protect DNA from oxidative damage. It also has the potential of playing a regulatory role in transcription. Nuclear ferritin represents a novel new outlook on ferritin functionality beyond its classical role as an iron storage molecule. Copyright 2010 Elsevier B.V. All rights reserved.

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

PubMed Central

Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

2015-01-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

Maresin 1, a Proresolving Lipid Mediator, Mitigates Carbon Tetrachloride-Induced Liver Injury in Mice

PubMed Central

Li, Ruidong; Wang, Yaxin; Zhao, Ende; Wu, Ke; Li, Wei; Shi, Liang; Wang, Di; Xie, Gengchen; Yin, Yuping; Deng, Meizhou; Zhang, Peng; Tao, Kaixiong

2016-01-01

Maresin 1 (MaR 1) was recently reported to have protective properties in several different animal models of acute inflammation by inhibiting inflammatory response. However, its function in acute liver injury is still unknown. To address this question, we induced liver injury in BALB/c mice with intraperitoneal injection of carbon tetrachloride with or without treatment of MaR 1. Our data showed that MaR 1 attenuated hepatic injury, oxidative stress, and lipid peroxidation induced by carbon tetrachloride, as evidenced by increased thiobarbituric acid reactive substances and reactive oxygen species levels were inhibited by treatment of MaR 1. Furthermore, MaR 1 increased activities of antioxidative mediators in carbon tetrachloride-treated mice liver. MaR 1 decreased indices of inflammatory mediators such as tumor necrosis factor-α, interleukin-6, interleukin-1β, monocyte chemotactic protein 1, myeloperoxidase, cyclooxygenase-2, and inducible nitric oxide synthase. Administration of MaR 1 inhibited activation of nuclear factor kappa B (NF-κb) and mitogen-activated protein kinases (MAPKs) in the liver of CCl4 treated mice. In conclusion, these results suggested the antioxidative, anti-inflammatory properties of MaR 1 in CCl4 induced liver injury. The possible mechanism is partly implicated in its abilities to inhibit ROS generation and activation of NF-κb and MAPK pathway. PMID:26881046

Radiation-induced cognitive dysfunction and cerebellar oxidative stress in mice: protective effect of alpha-lipoic acid.

PubMed

Manda, Kailash; Ueno, Megumi; Moritake, Takashi; Anzai, Kazunori

2007-02-12

Reactive oxygen species are implicated in neurodegeneration and cognitive disorders due to higher vulnerability of neuronal tissues. The cerebellum is recently reported to be involved in cognitive function. Therefore, present study aimed at investigating the role alpha-lipoic acid against radiation-induced oxidative stress and antioxidant status in cerebellum and its correlation with cognitive dysfunction. We observed spontaneous motor activities and spatial memory task of mice using pyroelectric infrared sensor and programmed video tracking system, respectively. Whole body X-irradiation (6 Gy) of mice substantially impaired the reference memory and motor activities of mice. However, acute intraperitoneal treatment of mice with alpha-lipoic acid prior to irradiation significantly attenuated such cognitive dysfunction. Alpha-lipoic acid pretreatment exerted a very high magnitude of protection against radiation-induced augmentation of protein carbonyls and thiobarbituric acid reactive substance (TBARS) in mice cerebellum. Further, radiation-induced deficit of total, nonprotein and protein-bound sulfhydryl (T-SH, NP-SH, PB-SH) contents of cerebellum and plasma ferric reducing power (FRAP) was also inhibited by alpha-lipoic acid pre-treatment. Moreover, alpha-lipoic acid treated mice showed an intact cytoarchitecture of cerebellum, higher counts of intact Purkinje cells and granular cells in comparison to untreated irradiated mice. Results clearly indicate that alpha-lipoic acid is potent neuroprotective antioxidant.

Blood Flow Modulation of Vascular Dynamics

PubMed Central

Lee, Juhyun; Sevag Packard, René R.; Hsiai, Tzung K.

2015-01-01

Purpose of review Blood flow is intimately linked with cardiovascular development, repair, and dysfunction. The current review will build on the fluid mechanical principle underlying hemodynamic shear forces, mechanotransduction, and metabolic effects. Recent findings Pulsatile flow produces both time- (∂τ /∂t)and spatial-varying shear stress (∂τ /∂x) to modulate vascular oxidative stress and inflammatory response with pathophysiological significance to atherosclerosis. The characteristics of hemodynamic shear forces; namely, steady laminar (∂τ /∂t= 0), pulsatile (PSS: unidirectional forward flow), and oscillatory shear stress (OSS: bidirectional with a near net 0 forward flow) modulate mechano-signal transduction to influence metabolic effects on vascular endothelial function. Atheroprotective PSS promotes anti-oxidant, anti-inflammatory, and anti-thrombotic responses, whereas atherogenic OSS induces NADPH oxidase–JNK signaling to increase mitochondrial superoxide production, protein degradation of manganese superoxide dismutase (MnSOD), and post-translational protein modifications of LDL particles in the disturbed flow-exposed regions of vasculature. In the era of tissue regeneration, shear stress has been implicated in re-activation of developmental genes; namely, Wnt and Notch signaling, for vascular development and repair. Summary Blood flow imparts a dynamic continuum from vascular development to repair. Augmentation of PSS confers atheroprotection and re-activation of developmental signaling pathways for regeneration. PMID:26218416

Cerebrosides from Sea Cucumber Protect Against Oxidative Stress in SAMP8 Mice and PC12 Cells.

PubMed

Che, Hongxia; Du, Lei; Cong, Peixu; Tao, Suyuan; Ding, Ning; Wu, Fengjuan; Xue, Changhu; Xu, Jie; Wang, Yuming

2017-04-01

Alzheimer's disease (AD) is a neurodegenerative disorder. Emerging evidence implicates β-amyloid (Aβ) plays a critical role in the progression of AD. In this study, we investigated the protective effect of cerebrosides obtained from sea cucumber against senescence-accelerated mouse prone 8 (SAMP8) mice in vivo. We also studied the effect of cerebrosides on Aβ-induced cytotoxicity on the rat pheochromocytoma cell (PC12) and the underlying molecular mechanisms. Cerebrosides ameliorated learning and memory deficits and the Aβ accumulation in demented mice, decreased the content of malondialdehyde (MDA), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG), 8-hydroxy-2'-deoxyguanosine (8-oxo-G), and nitric oxide (NO), and enhanced the superoxide dismutase (SOD) activity significantly. The neuroprotective effect of sea cucumber cerebrosides (SCC) was also verified in vitro: the cerebrosides increased the survival rate of PC12 cells, recovered the cellular morphology, downregulated the protein levels of Caspase-9, cleaved Caspase-3, total Caspase-3, and Bax, and upregulated the protein level of Bcl-2, revealing that cerebrosides could inhibit Aβ-induced cell apoptosis. The results showed the protective effect of SCC was regulated by the mitochondria-dependent apoptotic pathway. Our results provide a new approach to developing the marine organisms as functional foods for neuroprotection.