Redox proteomic evaluation of oxidative modification and recovery in a 3D reconstituted human skin tissue model exposed to UVB.

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Dyer, J M; Haines, S R; Thomas, A; Wang, W; Walls, R J; Clerens, S; Harland, D P

2017-04-01

Exposure to UV in humans resulting in sunburn triggers a complex series of events that are a mix of immediate and delayed damage mediation and healing. While studies on the effects of UV exposure on DNA damage and repair have been reported, changes in the oxidative modification of skin proteins are poorly understood at the molecular level, despite the important role played by structural proteins in skin tissue, and the effect of the integrity of these proteins on skin appearance and health. Proteomic molecular mapping of oxidation was here applied to try to enhance understanding of skin damage and recovery from oxidative damage and UVB exposure. A redox proteomic-based approach was applied to evaluating skin protein modification when exposed to varying doses of UVB after initial oxidative stress, via tracking changes in protein oxidation during the healing process in vitro using a full-thickness reconstituted human skin tissue model. Bioassays and structural evaluation confirmed that our cultured skin tissues underwent a normal physiological response to UVB exposure. A set of potential skin marker peptides was generated, for use in tracking skin protein oxidative modification. Exposure to UVB after thermal oxidative stress was found to result in higher levels of skin protein oxidation than a non-irradiated control for up to seven days after exposure. Recovery of the skin proteins from oxidative stress, as assessed by the overall protein oxidation levels, was found to be impaired by UVB exposure. Oxidative modification was largely observed in skin structural proteins. Exposure of skin proteins to UVB exacerbates oxidative damage to structural skin proteins, with higher exposure levels leading to increasingly impaired recovery from this damage. This has potential implications for the functional performance of the proteins and inter-related skin health and cosmetic appearance. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Advanced Oxidation Protein Products and Carbonylated Proteins as Biomarkers of Oxidative Stress in Selected Atherosclerosis-Mediated Diseases

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Bogna Gryszczyńska

2017-01-01

Full Text Available Objectives. The main question of this study was to evaluate the intensity of oxidative protein modification shown as advanced oxidation protein products (AOPP and carbonylated proteins, expressed as protein carbonyl content (C=O in abdominal aortic aneurysms (AAA, aortoiliac occlusive disease (AIOD, and chronic kidney disease (CKD. Design and Methods. The study was carried out in a group of 35 AAA patients and 13 AIOD patients. However, CKD patients were divided into two groups: predialysis (PRE included 50 patients or hemodialysis (HD consisted of 34 patients. AOPP and C=O were measured using colorimetric assay kit, while C-reactive protein concentration was measured by high-sensitivity assay (hsCRP. Results. The concentration of AOPP in both AAA and AIOD groups was higher than in PRE and HD groups according to descending order: AAA~AIOD > HD > PRE. The content of C=O was higher in the PRE group in comparison to AIOD and AAA according to the descending order: PRE~HD > AAA~AIOD. Conclusions. AAA, AIOD, and CKD-related atherosclerosis (PRE and HD contribute to the changes in the formation of AOPP and C=O. They may promote modification of proteins in a different way, probably due to the various factors that influence oxidative stress here.

Amides are novel protein modifications formed by physiological sugars.

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Glomb, M A; Pfahler, C

2001-11-09

The Maillard reaction, or nonenzymatic browning, proceeds in vivo, and the resulting protein modifications (advanced glycation end products) have been associated with various pathologies. Despite intensive research only very few structures have been established in vivo. We report here for the first time N(6)-[2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl]lysine (GOLA) and N(6)-glycoloyllysine (GALA) as prototypes for novel amide protein modifications produced by reducing sugars. Their identity was confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model reactions with N(alpha)-t-butoxycarbonyl-lysine showed that glyoxal and glycolaldehyde are immediate precursors, and reaction pathways are directly linked to N(epsilon)-carboxymethyllysine via glyoxal-imine structures. GOLA, the amide cross-link, and 1,3-bis(5-amino-5-carboxypentyl)imidazolium salt (GOLD), the imidazolium cross-link, share a common intermediate. The ratio of GOLA to GOLD is greater when glyoxal levels are low at constant lysine concentrations. GOLA and GALA formation from the Amadori product of glucose and lysine depends directly upon oxidation. With the advanced glycation end product inhibitors aminoguanidine and pyridoxamine we were able to dissect oxidative fragmentation of the Amadori product as a second mechanism of GOLA formation exactly coinciding with N(epsilon)-carboxymethyllysine synthesis. In contrast, the formation of GALA appears to depend solely upon glyoxal-imines. After enzymatic hydrolysis GOLA was found at 66 pmol/mg of brunescent lens protein. This suggests amide protein modifications as important markers of pathophysiological processes.

Oxidant/antioxidant balance in animal nutrition and health: the role of protein oxidation

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Pietro eCeli

2015-10-01

Full Text Available This review examines the role that oxidative stress, and protein oxidation in particular, plays in nutrition, metabolism and health of farm animals. The route by which redox homeostasis is involved in some important physiological functions and the implications of the impairment of oxidative status on animal health and diseases is also examined. Proteins have various and, at the same time, unique biological functions and their oxidation can result in structural changes and various functional modifications. Protein oxidation seems to be involved in pathological conditions such as respiratory diseases and parasitic infection; however some studies also suggest that protein oxidation plays a crucial role in the regulation of important physiological functions such as reproduction, nutrition, metabolism, lactation, gut health and neonatal physiology. As the characterization of the mechanisms by which oxidative stress may influence metabolism and health is attracting considerable scientific interest, the aim of this review is to present veterinary scientists and clinicians with various aspects of oxidative damage to proteins.

Protein and lipid oxidation affect the viscoelasticity of whey protein layers at the oil-water interface

NARCIS (Netherlands)

Berton-Carabin, Claire C.; Schroder, Anja; Rovalino-Cordova, Ana; Schroën, Karin; Sagis, Leonard

2016-01-01

Protein and lipid oxidation are prevailing issues that negatively affect the nutritional and sensory quality of food emulsions. It is probable that such oxidative modifications affect the functional properties of proteins, and in particular their ability to form densely packed, interconnected

Gamma irradiation effect on soy protein modification, protein - phenolic interaction and antioxidant activity in soybean

International Nuclear Information System (INIS)

Kumari, Sweta; Dahuja, Anil; Vinutha, T.; Singh, Bhupinder

2014-01-01

Soy protein is one of the most important sources of protein to feed the world population in the future. Consumption of soybean quality protein and their texture is dependent on the protein modification. In the present study, four soybean genotypes PL5039 (black), EC 472143 (black), Pusa 9814 (yellow) and SL525 (yellow), differing in their seed coat colour were gamma irradiated at 0.5,1.0, 2.0 and 5.0 kGy and the extent of protein modification and parameters affecting it viz. free phenolics, bound phenolics, lip oxygenase and antioxidant activity were analysed. Modifications of soybean proteins were investigated by chemical analysis and electrophoresis. The irradiation dose of 1.0 kGy showed decreased turbidity, protein oxidation, surface hydrophobicity but increased solubility and sulfhydryl and disulfide contents in all the genotypes. Further, SDS PAGE profile of treated soybean seeds revealed remarkable difference in electrophoretic bands as compared to the untreated seeds. Lipoxygense activity in all the genotypes decreased with increased exposure of gamma irradiation, which produced peroxide products that changes the structural characteristics of soy protein. Free phenolics, bound phenolics and total antioxidant activity measured in terms of FRAP in all the genotypes increased significantly at a dose of 2.0 kGy and it declined at a dose of 5.0 kGy. Antioxidant potential measured in terms of 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity showed an increasing trend with dose, indicating that radiation processing as a method of food preservation has a positive nutritional implication. Hence, it is suggested that, mild gamma irradiation upto 2.0 kGy may reduce the protein oxidation, enhance the antioxidant activity and improve the soybean protein quality compared to higher dose 5.0 kGy, which reduced the protein quality. (author)

Structural basis of protein oxidation resistance: a lysozyme study.

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Marion Girod

Full Text Available Accumulation of oxidative damage in proteins correlates with aging since it can cause irreversible and progressive degeneration of almost all cellular functions. Apparently, native protein structures have evolved intrinsic resistance to oxidation since perfectly folded proteins are, by large most robust. Here we explore the structural basis of protein resistance to radiation-induced oxidation using chicken egg white lysozyme in the native and misfolded form. We study the differential resistance to oxidative damage of six different parts of native and misfolded lysozyme by a targeted tandem/mass spectrometry approach of its tryptic fragments. The decay of the amount of each lysozyme fragment with increasing radiation dose is found to be a two steps process, characterized by a double exponential evolution of their amounts: the first one can be largely attributed to oxidation of specific amino acids, while the second one corresponds to further degradation of the protein. By correlating these results to the structural parameters computed from molecular dynamics (MD simulations, we find the protein parts with increased root-mean-square deviation (RMSD to be more susceptible to modifications. In addition, involvement of amino acid side-chains in hydrogen bonds has a protective effect against oxidation Increased exposure to solvent of individual amino acid side chains correlates with high susceptibility to oxidative and other modifications like side chain fragmentation. Generally, while none of the structural parameters alone can account for the fate of peptides during radiation, together they provide an insight into the relationship between protein structure and susceptibility to oxidation.

Oxidant/Antioxidant Balance in Animal Nutrition and Health: The Role of Protein Oxidation.

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Celi, Pietro; Gabai, Gianfranco

2015-01-01

This review examines the role that oxidative stress (OS), and protein oxidation in particular, plays in nutrition, metabolism, and health of farm animals. The route by which redox homeostasis is involved in some important physiological functions and the implications of the impairment of oxidative status on animal health and diseases is also examined. Proteins have various and, at the same time, unique biological functions and their oxidation can result in structural changes and various functional modifications. Protein oxidation seems to be involved in pathological conditions, such as respiratory diseases and parasitic infection; however, some studies also suggest that protein oxidation plays a crucial role in the regulation of important physiological functions, such as reproduction, nutrition, metabolism, lactation, gut health, and neonatal physiology. As the characterization of the mechanisms by which OS may influence metabolism and health is attracting considerable scientific interest, the aim of this review is to present veterinary scientists and clinicians with various aspects of oxidative damage to proteins.

Impaired metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes

DEFF Research Database (Denmark)

Baraibar, Martin; Hyzewicz, Janek; Rogowska-Wrzesinska, Adelina

2014-01-01

Accumulation of damaged macromolecules, including irreversibly oxidized proteins, is a hallmark of cellular and organismal ageing. Failure of protein homesotasis is a major contributor to the age-related accumulation of damaged proteins. In skeletal muscle, tissue maintenance and regeneration...... phenotype. In addition, these findings highlight the molecular mechanisms implicated in satellite cells dysfunction during ageing, paving the road for future therapeutic interventions aimed at preventing oxidative modifications of proteins and/or stimulating their elimination....

Protein Modifications as Manifestations of Hyperglycemic Glucotoxicity in Diabetes and Its Complications

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Hong Zheng

2016-01-01

Full Text Available Diabetes and its complications are hyperglycemic toxicity diseases. Many metabolic pathways in this array of diseases become aberrant, which is accompanied with a variety of posttranslational protein modifications that in turn reflect diabetic glucotoxicity. In this review, we summarize some of the most widely studied protein modifications in diabetes and its complications. These modifications include glycation, carbonylation, nitration, cysteine S-nitrosylation, acetylation, sumoylation, ADP-ribosylation, O-GlcNAcylation, and succination. All these posttranslational modifications can be significantly attributed to oxidative stress and/or carbon stress induced by diabetic redox imbalance that is driven by activation of pathways, such as the polyol pathway and the ADP-ribosylation pathway. Exploring the nature of these modifications should facilitate our understanding of the pathological mechanisms of diabetes and its associated complications.

Oxidative modifications, mitochondrial dysfunction, and impaired protein degradation in Parkinson's disease: how neurons are lost in the Bermuda triangle

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Malkus Kristen A

2009-06-01

Full Text Available Abstract While numerous hypotheses have been proposed to explain the molecular mechanisms underlying the pathogenesis of neurodegenerative diseases, the theory of oxidative stress has received considerable support. Although many correlations have been established and encouraging evidence has been obtained, conclusive proof of causation for the oxidative stress hypothesis is lacking and potential cures have not emerged. Therefore it is likely that other factors, possibly in coordination with oxidative stress, contribute to neuron death. Using Parkinson's disease (PD as the paradigm, this review explores the hypothesis that oxidative modifications, mitochondrial functional disruption, and impairment of protein degradation constitute three interrelated molecular pathways that execute neuron death. These intertwined events are the consequence of environmental exposure, genetic factors, and endogenous risks and constitute a "Bermuda triangle" that may be considered the underlying cause of neurodegenerative pathogenesis.

Protein oxidation in plant mitochondria as a stress indicator

DEFF Research Database (Denmark)

Møller, I.M.; Kristensen, B.K.

2004-01-01

oxidation of cysteine and methionine side chains is an important mechanism for regulating enzyme activity. Mitochondria from both mammalian and plant tissues contain a number of oxidised proteins, but the relative abundance of these post-translationally modified forms is as yet unknown......, as are the consequences of the modification for the properties and turnover time of the proteins. Specific proteins appear to be particularly vulnerable to oxidative carbonylation in the matrix of plant mitochondria; these include several enzymes of the Krebs cycle, glycine decarboxylase, superoxide dismutase and heat...... shock proteins. Plant mitochondria contain a number of different proteases, but their role in removing oxidatively damaged proteins is, as yet, unclear....

Exploring oxidative modifications of tyrosine

DEFF Research Database (Denmark)

Houée-Lévin, C; Bobrowski, K; Horakova, L

2015-01-01

residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different...... effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor...... residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation...

A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

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Patrizia Marinelli

2018-04-01

Full Text Available Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states. Keywords: Protein oxidation, Protein misfolding, Protein aggregation, Oxidative stress, Post-translational modification

Nitric Oxide-Mediated Posttranslational Modifications: Impacts at the Synapse

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Sophie A. Bradley

2016-01-01

Full Text Available Nitric oxide (NO is an important gasotransmitter molecule that is involved in numerous physiological processes throughout the nervous system. In addition to its involvement in physiological plasticity processes (long-term potentiation, LTP; long-term depression, LTD which can include NMDAR-mediated calcium-dependent activation of neuronal nitric oxide synthase (nNOS, new insights into physiological and pathological consequences of nitrergic signalling have recently emerged. In addition to the canonical cGMP-mediated signalling, NO is also implicated in numerous pathways involving posttranslational modifications. In this review we discuss the multiple effects of S-nitrosylation and 3-nitrotyrosination on proteins with potential modulation of function but limit the analyses to signalling involved in synaptic transmission and vesicular release. Here, crucial proteins which mediate synaptic transmission can undergo posttranslational modifications with either pre- or postsynaptic origin. During normal brain function, both pathways serve as important cellular signalling cascades that modulate a diverse array of physiological processes, including synaptic plasticity, transcriptional activity, and neuronal survival. In contrast, evidence suggests that aging and disease can induce nitrosative stress via excessive NO production. Consequently, uncontrolled S-nitrosylation/3-nitrotyrosination can occur and represent pathological features that contribute to the onset and progression of various neurodegenerative diseases, including Parkinson’s, Alzheimer’s, and Huntington’s.

Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

KAUST Repository

Marondedze, Claudius

2013-01-05

Background: Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins. Findings. Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus. Conclusions: Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated. 2013 Marondedze et al.; licensee BioMed Central Ltd.

Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation

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Background: Reversible posttranslational protein modifications such as phosphorylation of Ser/Thr/Tyr and Met oxidation are critical for both metabolic regulation and cellular signalling. Although these modifications are typically studied individually, herein we describe the potential for cross-talk...

Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

Energy Technology Data Exchange (ETDEWEB)

Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun

2017-01-01

Protein cysteine thiols play a crucial role in redox signaling, regulation of enzymatic activity and protein function, and maintaining redox homeostasis in living systems. The unique chemical reactivity of thiol groups makes cysteine susceptible to oxidative modifications by reactive oxygen and nitrogen species to form a broad array of reversible and irreversible protein post-translational modifications (PTMs). The reversible modifications in particular are one of the major components of redox signaling and are involved in regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in health and diseases has been increasingly recognized. Herein, we review the recent advances of quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including the general considerations of sample processing, various chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for addressing specific biological questions. Although some technological limitations remain, redox proteomics is paving the way towards a better understanding of redox signaling and regulation in human health and diseases.

Acrolein, A Reactive Product of Lipid Peroxidation, Induces Oxidative Modification of Cytochrome c

Energy Technology Data Exchange (ETDEWEB)

Kang, Jung Hoon [Cheongju Univ., Cheongju (Korea, Republic of)

2013-11-15

Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). In Alzheimer's brain, ACR was found to be elevated in hippocampus and temporal cortex where oxidative stress is high. In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with ACR. When cytochrome c was incubated with ACR, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in ACR-treated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the ACR-mediated cytochrome c modification and carbonyl compound formation. Our data demonstrate that oxidative damage of cytochrome c by ACR might induce disruption of cyotochrome c structure and iron mishandling as a contributing factor to the pathology of AD.

Acrolein, A Reactive Product of Lipid Peroxidation, Induces Oxidative Modification of Cytochrome c

International Nuclear Information System (INIS)

Kang, Jung Hoon

2013-01-01

Acrolein (ACR) is a well-known carbonyl toxin produced by lipid peroxidation of polyunsaturated fatty acids, which is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). In Alzheimer's brain, ACR was found to be elevated in hippocampus and temporal cortex where oxidative stress is high. In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with ACR. When cytochrome c was incubated with ACR, protein aggregation increased in a dose-dependent manner. The formation of carbonyl compounds and the release of iron were obtained in ACR-treated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the ACR-mediated cytochrome c modification and carbonyl compound formation. Our data demonstrate that oxidative damage of cytochrome c by ACR might induce disruption of cyotochrome c structure and iron mishandling as a contributing factor to the pathology of AD

Clarithromycin, trimethoprim, and penicillin and oxidative nucleic acid modifications in humans

DEFF Research Database (Denmark)

Larsen, Emil List; Cejvanovic, Vanja; Kjaer, Laura Kofoed

2017-01-01

, phenoxymethylpenicillin (penicillin V), or placebo. Oxidative modifications were measured as 24-h urinary excretion of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and plasma levels of malondialdehyde before and after treatment as a measurement of DNA oxidation, RNA oxidation.......7% (95% CI: 5.8–37.6%), but did not influence urinary excretion of 8-oxoGuo. Penicillin V did not influence urinary excretion of 8-oxodG or 8-oxoGuo. None of the antibiotic drugs influenced plasma levels of malondialdehyde. Conclusion Clarithromycin significantly increases oxidative nucleic acid...... modifications. Increased oxidative modifications might explain some of clarithromycin's known adverse reactions. Trimethoprim significantly lowers DNA oxidation but not RNA oxidation. Penicillin V had no effect on oxidative nucleic acid modifications....

Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.

Science.gov (United States)

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.

Mass Spectrometry-Based Methods for Identifying Oxidized Proteins in Disease: Advances and Challenges

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Ivan Verrastro

2015-04-01

Full Text Available Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease.

Role of Oxidative Stress in Epigenetic Modification in Endometriosis.

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Ito, Fuminori; Yamada, Yuki; Shigemitsu, Aiko; Akinishi, Mika; Kaniwa, Hiroko; Miyake, Ryuta; Yamanaka, Shoichiro; Kobayashi, Hiroshi

2017-11-01

Aberrant DNA methylation and histone modification are associated with an increased risk of reproductive disorders such as endometriosis. However, a cause-effect relationship between epigenetic mechanisms and endometriosis development has not been fully determined. This review provides current information based on oxidative stress in epigenetic modification in endometriosis. This article reviews the English-language literature on epigenetics, DNA methylation, histone modification, and oxidative stress associated with endometriosis in an effort to identify epigenetic modification that causes a predisposition to endometriosis. Oxidative stress, secondary to the influx of hemoglobin, heme, and iron during retrograde menstruation, is involved in the expression of CpG demethylases, ten-eleven translocation, and jumonji (JMJ). Ten-eleven translocation and JMJ recognize a wide range of endogenous DNA methyltransferases (DNMTs). The increased expression levels of DNMTs may be involved in the subsequent downregulation of the decidualization-related genes. This review supports the hypothesis that there are at least 2 distinct phases of epigenetic modification in endometriosis: the initial wave of iron-induced oxidative stress would be followed by the second big wave of epigenetic modulation of endometriosis susceptibility genes. We summarize the recent advances in our understanding of the underlying epigenetic mechanisms focusing on oxidative stress in endometriosis.

Assessment of protein modifications in liver of rats under chronic treatment with paracetamol (acetaminophen) using two complementary mass spectrometry-based metabolomic approaches.

Science.gov (United States)

Mast, Carole; Lyan, Bernard; Joly, Charlotte; Centeno, Delphine; Giacomoni, Franck; Martin, Jean-François; Mosoni, Laurent; Dardevet, Dominique; Pujos-Guillot, Estelle; Papet, Isabelle

2015-04-29

Liver protein can be altered under paracetamol (APAP) treatment. APAP-protein adducts and other protein modifications (oxidation/nitration, expression) play a role in hepatotoxicity induced by acute overdoses, but it is unknown whether liver protein modifications occur during long-term treatment with non-toxic doses of APAP. We quantified APAP-protein adducts and assessed other protein modifications in the liver from rats under chronic (17 days) treatment with two APAP doses (0.5% or 1% of APAP in the diet w/w). A targeted metabolomic method was validated and used to quantify APAP-protein adducts as APAP-cysteine adducts following proteolytic hydrolysis. The limit of detection was found to be 7ng APAP-cysteine/mL hydrolysate i.e. an APAP-Cys to tyrosine ratio of 0.016‰. Other protein modifications were assessed on the same protein hydrolysate by untargeted metabolomics including a new strategy to process the data and identify discriminant molecules. These two complementary mass spectrometry (MS)-based metabolic approaches enabled the assessment of a wide range of protein modifications induced by chronic treatment with APAP. APAP-protein adducts were detected even in the absence of glutathione depletion and hepatotoxicity, i.e. in the 0.5% APAP group, and increased by 218% in the 1% APAP group compared to the 0.5% APAP group. At the same time, the untargeted metabolomic method revealed a decrease in the binding of cysteine, cysteinyl-glycine and GSH to thiol groups of protein cysteine residues, an increase in the oxidation of tryptophan and proline residues and a modification in protein expression. This wide range of modifications in liver proteins occurred in rats under chronic treatment with APAP that did not induce hepatotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

Oxidative modification of ferritin induced by methylglyoxal

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Sung Ho An

2012-03-01

Full Text Available Methylglyoxal (MG was identified as an intermediate innon-enzymatic glycation and increased levels were reported inpatients with diabetes. In this study, we evaluated the effects ofMG on the modification of ferritin. When ferritin wasincubated with MG, covalent crosslinking of the proteinincreased in a time- and MG dose-dependent manner.Reactive oxygen species (ROS scavengers, N-acetyl-L-cysteineand thiourea suppressed the MG-mediated ferritinmodification. The formation of dityrosine was observed inMG-mediated ferritin aggregates and ROS scavengers inhibitedthe formation of dityrosine. During the reaction betweenferritin and MG, the generation of ROS was increased as afunction of incubation time. These results suggest that ROSmay play a role in the modification of ferritin by MG. Thereaction between ferritin and MG led to the release of ironions from the protein. Ferritin exposure to MG resulted in aloss of arginine, histidine and lysine residues. It was assumedthat oxidative damage to ferritin caused by MG may induce anincrease in the iron content in cells, which is deleterious tocells. This mechanism, in part, may provide an explanation orthe deterioration of organs under diabetic conditions. [BMBreports 2012; 45(3: 147-152

Modification in oxidative processes in muscle tissues exposed to laser- and light-emitting diode radiation.

Science.gov (United States)

Monich, Victor A; Bavrina, Anna P; Malinovskaya, Svetlana L

2018-01-01

Exposure of living tissues to high-intensity red or near-infrared light can produce the oxidative stress effects both in the target zone and adjacent ones. The protein oxidative modification (POM) products can be used as reliable and early markers of oxidative stress. The contents of modified proteins in the investigated specimens can be evaluated by the 2,4-dinitrophenylhydrazine assay (the DNPH assay). Low-intensity red light is able to decrease the activity of oxidative processes and the DNPH assay data about the POM products in the biological tissues could show both an oxidative stress level and an efficiency of physical agent protection against the oxidative processes. Two control groups of white rats were irradiated by laser light, the first control group by red light and the second one by near-infrared radiation (NIR).Two experimental groups were consequently treated with laser and red low-level light-emitting diode radiation (LED). One of them was exposed to red laser light + LED and the other to NIR + LED. The fifth group was intact. Each group included ten animals. The effect of laser light was studied by methods of protein oxidative modifications. We measured levels of both induced and spontaneous POM products by the DNPH assay. The dramatic increase in levels of POM products in the control group samples when compared with the intact group data as well as the sharp decrease in the POM products in the experimental groups treated with LED low-level light were statistically significant (p ≤ 0.05). Exposure of skeletal muscles to high-intensity red and near-infrared laser light causes oxidative stress that continues not less than 3 days. The method of measurement of POM product contents by the DNPH assay is a reliable test of an oxidative process rate. Red low-intensity LED radiation can provide rehabilitation of skeletal muscle tissues treated with high-intensity laser light.

Role of protein and mRNA oxidation in seed dormancy and germination

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hayat eel-maarouf-bouteau

2013-04-01

Full Text Available Reactive oxygen species (ROS are key players in the regulation of seed germination and dormancy. Although their regulated accumulation is a prerequisite for germination, the cellular basis of their action remains unknown, but very challenging to elucidate due to the lack of specificity of these compounds that can potentially react with all biomolecules. Among these, nucleic acids and proteins are very prone to oxidative damage. RNA is highly sensitive to oxidation because of its single-stranded structure and the absence of a repair system. Oxidation of mRNAs induces their decay through processing bodies or results in the synthesis of aberrant proteins through altered translation. Depending on the oxidized amino acid, ROS damage of proteins can be irreversible (i.e. carbonylation thus triggering the degradation of the oxidized proteins by the cytosolic 20S proteasome or can be reversed through the action of thioredoxins, peroxiredoxins or glutaredoxins (cysteine oxidation or by methionine sulfoxide reductase (methionine oxidation. Seed dormancy alleviation in the dry state, referred to as after-ripening, requires both selective mRNA oxidation and protein carbonylation. Similarly, seed imbibition of non-dormant seeds is associated with targeted oxidation of a subset of proteins. Altogether, these specific features testify that such oxidative modifications play important role in commitment of the cellular functioning toward germination completion.

Influence of low- and high-dose radioiodine therapy on oxidative modification of fibrinogen

International Nuclear Information System (INIS)

Schweeger-Exeli, I.J.

2001-10-01

Fibrinogen plays a central role in the course of thrombosis and hemostasis. It is soluble in blood and tissue extracts and transformed into the insoluble fibrin network structure in the presence of thrombin. Fibrinogen in circulating blood consists of a population of slightly different molecules with a half-life of 3.5-4.5 days. Various environmental conditions may cause different types of modifications of the molecule leading to a different functional behavior. Introduction of carbonyl groups in amino acid side chains is known as a marker for protein oxidation. Radioiodine therapy, applied in patients suffering from hyperthyroidism or differentiated thyroid carcinoma, may cause an oxidative modification of fibrinogen by formation of free radicals in blood exposed to the radioactive agent 131I. The topic of my thesis was i. to develop a simple and not time consuming method for isolation of fibrinogen from small volumes of human plasma (∼ 6ml), ii. to assess, whether radioiodine therapy causes detectable introduction of carbonyl groups into the fibrinogen molecule, and iii. to analyze an association between thyroid hormone function, fibrinogen levels and protein oxidation by means of carbonyl content. Purification of fibrinogen from human plasma was possible by three different methods (ammonium sulphate/ethanol; glycine/ethanol; glycine). Plasma levels of fibrinogen (Clauss method) and protein carbonyl group content (2,4-DNPH - assay) were determined before and after radioiodine therapy. The results demonstrate a significant increase (p = 0.05) in carbonyl content of human fibrinogen in cancer patients treated with 131I. However, in patients with diagnosed hyperthyroidism values were not significantly altered. In carcinoma patients, baseline fT4 levels and the relative increase in carbonyl content of fibrinogen after radioiodine therapy were correlated (r = 0.83; p 0.005), whereas no such correlation was found in patients with hyperthyroidism. Plasma fibrinogen

Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

Energy Technology Data Exchange (ETDEWEB)

Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

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 human 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.

Protein oxidation in aquatic foods

DEFF Research Database (Denmark)

Baron, Caroline P.

2014-01-01

The chapter discusses general considerations about protein oxidation and reviews the mechanisms involved in protein oxidation and consequences of protein oxidation on fish proteins. It presents two case studies, the first deals with protein and lipid oxidation in frozen rainbow trout......, and the second with oxidation in salted herring. The mechanisms responsible for initiation of protein oxidation are unclear, but it is generally accepted that free radical species initiating lipid oxidation can also initiate protein oxidation. The chapter focuses on interaction between protein and lipid...... oxidation. The protein carbonyl group measurement is the widely used method for estimating protein oxidation in foods and has been used in fish muscle. The chapter also talks about the impact of protein oxidation on protein functionality, fish muscle texture, and food nutritional value. Protein oxidation...

Chromatin proteins and modifications as drug targets

DEFF Research Database (Denmark)

Helin, Kristian; Dhanak, Dashyant

2013-01-01

A plethora of groundbreaking studies have demonstrated the importance of chromatin-associated proteins and post-translational modifications of histones, proteins and DNA (so-called epigenetic modifications) for transcriptional control and normal development. Disruption of epigenetic control...... is a frequent event in disease, and the first epigenetic-based therapies for cancer treatment have been approved. A generation of new classes of potent and specific inhibitors for several chromatin-associated proteins have shown promise in preclinical trials. Although the biology of epigenetic regulation...

Protein covalent modification by biologically active quinones

Directory of Open Access Journals (Sweden)

MIROSLAV J. GASIC

2004-11-01

Full Text Available The avarone/avarol quinone/hydroquinone couple shows considerable antitumor activity. In this work, covalent modification of b-lactoglobulin by avarone and its derivatives as well as by the synthetic steroidal quinone 2,5(10-estradiene-1,4,17-trione and its derivatives were studied. The techniques for studying chemical modification of b-lactoglobulin by quinones were: UV/Vis spectrophotometry, SDS PAGE and isoelectrofocusing. SDS PAGE results suggest that polymerization of the protein occurs. It could be seen that the protein of 18 kD gives the bands of 20 kD, 36 kD, 40 kD, 45 kD, 64 kD and 128 kD depending on modification agent. The shift of the pI of the protein (5.4 upon modification toward lower values (from pI 5.0 to 5.3 indicated that lysine amino groups are the principal site of the reaction of b-lactoglobulin with the quinones.

Laser- and UV-assisted modification of polystyrene surfaces for control of protein adsorption and cell adhesion

International Nuclear Information System (INIS)

Pfleging, Wilhelm; Torge, Maika; Bruns, Michael; Trouillet, Vanessa; Welle, Alexander; Wilson, Sandra

2009-01-01

An appropriate choice of laser and process parameters enables new approaches for the fabrication of polymeric lab-on-chip devices with integrated functionalities. We will present our current research results in laser-assisted modification of polystyrene (PS) with respect to the fabrication of polymer devices for cell culture applications. For this purpose laser micro-patterning of PS and subsequent surface functionalization was investigated as function of laser and process parameters. A high power ArF-excimer laser radiation source with a pulse length of 19 ns as well as a high repetition ArF-excimer laser source with a pulse length of 5 ns were used in order to study the influence of laser pulse length on laser-induced surface oxidation. The change in surface chemistry was characterized by X-ray photoelectron spectroscopy and contact angle measurements. The difference between laser-assisted modification versus UV-lamp assisted modification was investigated. A photolytic activation of specific areas of the polymer surface and subsequent oxidization in oxygen or ambient air leads to a chemically modified polymer surface bearing carboxylic acid groups well-suited for controlled competitive protein adsorption or protein immobilization. Finally, distinct areas for cell growth and adhesion are obtained

Surface modification of protein enhances encapsulation in chitosan nanoparticles

Science.gov (United States)

Koyani, Rina D.; Andrade, Mariana; Quester, Katrin; Gaytán, Paul; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

2018-04-01

Chitosan nanoparticles have a huge potential as nanocarriers for environmental and biomedical purposes. Protein encapsulation in nano-sized chitosan provides protection against inactivation, proteolysis, and other alterations due to environmental conditions, as well as the possibility to be targeted to specific tissues by ligand functionalization. In this work, we demonstrate that the chemical modification of the protein surface enhances the protein loading in chitosan nanocarriers. Encapsulation of green fluorescent protein and the cytochrome P450 was studied. The increase of electrostatic interactions between the free amino groups of chitosan and the increased number of free carboxylic groups in the protein surface enhance the protein loading, protein retention, and, thus, the enzymatic activity of chitosan nanoparticles. The chemical modification of protein surface with malonic acid moieties reduced drastically the protein isoelectric point increasing the protein interaction with the polycationic biomaterial and chitosan. The chemical modification of protein does not alter the morphology of chitosan nanoparticles that showed an average diameter of 18 nm, spheroidal in shape, and smooth surfaced. The strategy of chemical modification of protein surface, shown here, is a simple and efficient technique to enhance the protein loading in chitosan nanoparticles. This technique could be used for other nanoparticles based on polycationic or polyanionic materials. The increase of protein loading improves, doubtless, the performance of protein-loaded chitosan nanoparticles for biotechnological and biomedical applications.

Exploring the diversity of protein modifications: special bacterial phosphorylation systems

DEFF Research Database (Denmark)

Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

2016-01-01

Protein modifications not only affect protein homeostasis but can also establish new cellular protein functions and are important components of complex cellular signal sensing and transduction networks. Among these post-translational modifications, protein phosphorylation represents the one that ...

Analysis of the post-translational modifications of the individual amino acids in lens proteins which were induced by aging and irradiation

International Nuclear Information System (INIS)

Fujii, Noriko; Kim, Ingu; Saito, Takeshi; Takata, Takumi

2017-01-01

The eye lens is a transparent organ that functions to focus light and images on the retina. The transparency and high refraction of the lens are maintained by the function of α-, β- and γ-crystallins. These long-lived proteins are subject to various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, which occur gradually during the aging process. Such modifications, which are generated by UV light and oxidative stress, decrease crystallin solubility and lens transparency, and ultimately lead to the development of age-related cataracts. Here, we irradiated young rat lenses with γ-rays (5-500 Gy) and extracted the water-soluble (WS) and insoluble (WI) protein fractions. The WS and WI lens proteins were digested with trypsin, and the resulting peptides were analyzed by one-shot LC-MS/MS to determine the specific sites of oxidation of methionine and tryptophan, deamidation of asparagine and glutamine, and isomerization of aspartyl in rat α- and β-crystallins in the WS and WI fractions. Oxidation and deamidation occurred in several crystallins after irradiation at more than, respectively, 50 Gy and 5 Gy; however, isomerization did not occur in any crystallin even after exposure to 500 Gy of irradiation. The number of oxidation and deamidation sites was much higher in the WI than in the WS fraction. Furthermore, the oxidation and deamidation sites in rat crystallins resemble those reported in crystallins from human age-related cataracts. Thus, this study on post-translational modifications of crystallins induced by ionizing irradiation may provide useful information relevant to the formation of human age-related cataracts. (author)

Methionine sulfoxides in serum proteins as potential clinical biomarkers of oxidative stress

OpenAIRE

Satoko Suzuki; Yoshio Kodera; Tatsuya Saito; Kazumi Fujimoto; Akari Momozono; Akinori Hayashi; Yuji Kamata; Masayoshi Shichiri

2016-01-01

Oxidative stress contributes to the pathophysiology of a variety of diseases, and circulating biomarkers of its severity remains a topic of great interest for researchers. Our peptidomic strategy enables accurate and reproducible analysis of circulating proteins/peptides with or without post-translational modifications. Conventional wisdom holds that hydrophobic methionines exposed to an aqueous environment or experimental handling procedures are vulnerable to oxidation. However, we show that...

Protein Carbamylation: A Marker Reflecting Increased Age-Related Cell Oxidation

Directory of Open Access Journals (Sweden)

Julia Carracedo

2018-05-01

Full Text Available Carbamylation is a post-translational modification of proteins that may partake in the oxidative stress-associated cell damage, and its increment has been recently proposed as a “hallmark of aging”. The molecular mechanisms associated with aging are related to an increased release of free radicals. We have studied whether carbamylated proteins from the peripheral blood of healthy subjects are related to oxidative damage and aging, taking into account the gender and the immune profile of the subjects. The study was performed in healthy human volunteers. The detection of protein carbamylation and malondialdehyde (MDA levels was evaluated using commercial kits. The immune profile was calculated using parameters of immune cell function. The results show that the individuals from the elderly group (60–79 years old have increased carbamylated protein and MDA levels. When considered by gender, only men between 60 and 79 years old showed significantly increased carbamylated proteins and MDA levels. When those subjects were classified by their immune profile, the carbamylated protein levels were higher in those with an older immune profile. In conclusion, the carbamylation of proteins in peripheral blood is related to age-associated oxidative damage and to an aging functional immunological signature. Our results suggest that carbamylated proteins may play an important role at the cellular level in the aging process.

[Free radical modification of proteins in brain structure of Sprague-Dawley rats and some behaviour indicators after prenatal stress].

Science.gov (United States)

V'iushina, A V; Pritvorova, A V; Flerov, M A

2012-08-01

We studied the influence of late prenatal stress on free radical oxidation processes in Sprague-Dawley rats cortex, striatum, hippocampus, hypothalamus proteins. It was shown that after prenatal stress most changes were observed in hypothalamus and hippocampus. It was shown that in hypothalamus spontaneous oxidation level increased, but level of induced oxidation decreased, the opposite changes were found in hippocampus. Simultaneously minor changes of protein modification were observed in cortex and striatum. It was shown that prenatal stress changed both correlation of proteins free radical oxidation in studied structures and values of these data regarding to control. In test of "open field" motor activity in rats after prenatal stress decreased and time of freezing and grooming increased; opposite, in T-labyrinth motor activity and time of grooming in rats after prenatal stress increased, but time of freezing decreased.

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

Science.gov (United States)

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.

Soy protein modification: A review

Directory of Open Access Journals (Sweden)

Barać Miroljub B.

2004-01-01

Full Text Available Soy protein products such as flour, concentrates and isolates are used in food formulation because of their functionality, nutritional value and low cost. To obtain their optimal nutritive and functional properties as well as desirable flavor different treatments are used. Soybean proteins can be modified by physical, chemical and enzymatic treatments. Different thermal treatments are most commonly used, while the most appropriate way of modifying soy proteins from the standpoint of safety is their limited proteolysis. These treatments cause physical and chemical changes that affect their functional properties. This review discusses three principal methods used for modification of soy protein products, their effects on dominant soy protein properties and some biologically active compounds.

BioJava-ModFinder: identification of protein modifications in 3D structures from the Protein Data Bank.

Science.gov (United States)

Gao, Jianjiong; Prlic, Andreas; Bi, Chunxiao; Bluhm, Wolfgang F; Dimitropoulos, Dimitris; Xu, Dong; Bourne, Philip E; Rose, Peter W

2017-07-01

We developed a new software tool, BioJava-ModFinder, for identifying protein modifications observed in 3D structures archived in the Protein Data Bank (PDB). Information on more than 400 types of protein modifications were collected and curated from annotations in PDB, RESID, and PSI-MOD. We divided these modifications into three categories: modified residues, attachment modifications, and cross-links. We have developed a systematic method to identify these modifications in 3D protein structures. We have integrated this package with the RCSB PDB web application and added protein modification annotations to the sequence diagram and structure display. By scanning all 3D structures in the PDB using BioJava-ModFinder, we identified more than 30 000 structures with protein modifications, which can be searched, browsed, and visualized on the RCSB PDB website. BioJava-ModFinder is available as open source (LGPL license) at ( https://github.com/biojava/biojava/tree/master/biojava-modfinder ). The RCSB PDB can be accessed at http://www.rcsb.org . pwrose@ucsd.edu. © The Author 2017. Published by Oxford University Press.

Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids.

Science.gov (United States)

Iyer, Lakshminarayan M; Tahiliani, Mamta; Rao, Anjana; Aravind, L

2009-06-01

Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase super-family, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a

Covalent modification and exfoliation of graphene oxide using ferrocene

Science.gov (United States)

Avinash, M. B.; Subrahmanyam, K. S.; Sundarayya, Y.; Govindaraju, T.

2010-09-01

Large scale preparation of single-layer graphene and graphene oxide is of great importance due to their potential applications. We report a simple room temperature method for the exfoliation of graphene oxide using covalent modification of graphene oxide with ferrocene to obtain single-layer graphene oxide sheets. The samples were characterized by FESEM, HRTEM, AFM, EDAX, FT-IR, Raman and Mössbauer spectroscopic studies. HRTEM micrograph of the covalently modified graphene oxide showed increased interlayer spacing of ~2.4 nm due to ferrocene intercalation. The presence of single-layer graphene oxide sheets were confirmed by AFM studies. The covalently modified ferrocene-graphene oxide composite showed interesting magnetic behavior.Large scale preparation of single-layer graphene and graphene oxide is of great importance due to their potential applications. We report a simple room temperature method for the exfoliation of graphene oxide using covalent modification of graphene oxide with ferrocene to obtain single-layer graphene oxide sheets. The samples were characterized by FESEM, HRTEM, AFM, EDAX, FT-IR, Raman and Mössbauer spectroscopic studies. HRTEM micrograph of the covalently modified graphene oxide showed increased interlayer spacing of ~2.4 nm due to ferrocene intercalation. The presence of single-layer graphene oxide sheets were confirmed by AFM studies. The covalently modified ferrocene-graphene oxide composite showed interesting magnetic behavior. Electronic supplementary information (ESI) available: Magnetic data; AFM images; TEM micrographs; and Mössbauer spectroscopic data. See DOI: 10.1039/c0nr00024h

RNA modifications by oxidation

DEFF Research Database (Denmark)

Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

2012-01-01

to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

Site-selective protein-modification chemistry for basic biology and drug development.

Science.gov (United States)

Krall, Nikolaus; da Cruz, Filipa P; Boutureira, Omar; Bernardes, Gonçalo J L

2016-02-01

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

Specific histone modification responds to arsenic-induced oxidative stress

Energy Technology Data Exchange (ETDEWEB)

Ma, Lu [Department of Toxicology, Guangzhou Key Laboratory of Environmental Pollution and Health Risk Assessment, School of Public Health, Sun Yat-sen University, Guangzhou (China); Li, Jun [Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Department of Toxicology, School of Public Health, Guizhou Medical University, Guiyang, Guizhou (China); Zhan, Zhengbao; Chen, Liping; Li, Daochuan; Bai, Qing; Gao, Chen; Li, Jie; Zeng, Xiaowen; He, Zhini; Wang, Shan; Xiao, Yongmei [Department of Toxicology, Guangzhou Key Laboratory of Environmental Pollution and Health Risk Assessment, School of Public Health, Sun Yat-sen University, Guangzhou (China); Chen, Wen, E-mail: chenwen@mail.sysu.edu.cn [Department of Toxicology, Guangzhou Key Laboratory of Environmental Pollution and Health Risk Assessment, School of Public Health, Sun Yat-sen University, Guangzhou (China); Zhang, Aihua, E-mail: aihuagzykd@163.com [Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Department of Toxicology, School of Public Health, Guizhou Medical University, Guiyang, Guizhou (China)

2016-07-01

To explore whether specific histone modifications are associated with arsenic-induced oxidative damage, we recruited 138 arsenic-exposed and arsenicosis subjects from Jiaole Village, Xinren County of Guizhou province, China where the residents were exposed to arsenic from indoor coal burning. 77 villagers from Shang Batian Village that were not exposed to high arsenic coal served as the control group. The concentrations of urine and hair arsenic in the arsenic-exposure group were 2.4-fold and 2.1-fold (all P < 0.001) higher, respectively, than those of the control group. Global histone modifications in human peripheral lymphocytes (PBLCs) were examined by ELISA. The results showed that altered global levels of H3K18ac, H3K9me2, and H3K36me3 correlated with both urinary and hair-arsenic levels of the subjects. Notably, H3K36me3 and H3K18ac modifications were associated with urinary 8-OHdG (H3K36me3: β = 0.16; P = 0.042, H3K18ac: β = − 0.24; P = 0.001). We also found that the modifications of H3K18ac and H3K36me3 were enriched in the promoters of oxidative stress response (OSR) genes in human embryonic kidney (HEK) cells and HaCaT cells, providing evidence that H3K18ac and H3K36me3 modifications mediate transcriptional regulation of OSR genes in response to NaAsO{sub 2} treatment. Particularly, we found that reduced H3K18ac modification correlated with suppressed expression of OSR genes in HEK cells with long term arsenic treatment and in PBLCs of all the subjects. Taken together, we reveal a critical role for specific histone modification in response to arsenic-induced oxidative damage. - Highlights: • H3K18ac, H3K9me2 and H3K36me3 were associated with arsenic exposed levels. • H3K18ac and H3K36me3 were correlated with oxidative damage induced by arsenic. • H3K18ac and H3K36me3 might involve in transcriptional regulation of OSR genes. • Dysregulation of H3K18ac and H3K36me3 might be biomarkers of arsenic toxicity.

Specific histone modification responds to arsenic-induced oxidative stress

International Nuclear Information System (INIS)

Ma, Lu; Li, Jun; Zhan, Zhengbao; Chen, Liping; Li, Daochuan; Bai, Qing; Gao, Chen; Li, Jie; Zeng, Xiaowen; He, Zhini; Wang, Shan; Xiao, Yongmei; Chen, Wen; Zhang, Aihua

2016-01-01

To explore whether specific histone modifications are associated with arsenic-induced oxidative damage, we recruited 138 arsenic-exposed and arsenicosis subjects from Jiaole Village, Xinren County of Guizhou province, China where the residents were exposed to arsenic from indoor coal burning. 77 villagers from Shang Batian Village that were not exposed to high arsenic coal served as the control group. The concentrations of urine and hair arsenic in the arsenic-exposure group were 2.4-fold and 2.1-fold (all P < 0.001) higher, respectively, than those of the control group. Global histone modifications in human peripheral lymphocytes (PBLCs) were examined by ELISA. The results showed that altered global levels of H3K18ac, H3K9me2, and H3K36me3 correlated with both urinary and hair-arsenic levels of the subjects. Notably, H3K36me3 and H3K18ac modifications were associated with urinary 8-OHdG (H3K36me3: β = 0.16; P = 0.042, H3K18ac: β = − 0.24; P = 0.001). We also found that the modifications of H3K18ac and H3K36me3 were enriched in the promoters of oxidative stress response (OSR) genes in human embryonic kidney (HEK) cells and HaCaT cells, providing evidence that H3K18ac and H3K36me3 modifications mediate transcriptional regulation of OSR genes in response to NaAsO 2 treatment. Particularly, we found that reduced H3K18ac modification correlated with suppressed expression of OSR genes in HEK cells with long term arsenic treatment and in PBLCs of all the subjects. Taken together, we reveal a critical role for specific histone modification in response to arsenic-induced oxidative damage. - Highlights: • H3K18ac, H3K9me2 and H3K36me3 were associated with arsenic exposed levels. • H3K18ac and H3K36me3 were correlated with oxidative damage induced by arsenic. • H3K18ac and H3K36me3 might involve in transcriptional regulation of OSR genes. • Dysregulation of H3K18ac and H3K36me3 might be biomarkers of arsenic toxicity.

Nitric oxide-related species-induced protein oxidation: reversible, irreversible, and protective effects on enzyme function of papain.

Science.gov (United States)

Väänänen, Antti J; Kankuri, Esko; Rauhala, Pekka

2005-04-15

Protein oxidation, irreversible modification, and inactivation may play key roles in various neurodegenerative disorders. Therefore, we studied the effects of the potentially in vivo occurring nitric oxide-related species on two different markers of protein oxidation: protein carbonyl generation on bovine serum albumine (BSA) and loss of activity of a cysteine-dependent protease, papain, in vitro by using Angeli's salt, papanonoate, SIN-1, and S-nitrosoglutathione (GSNO) as donors of nitroxyl, nitric oxide, peroxynitrite, and nitrosonium ions, respectively. Angeli's salt, SIN-1, and papanonoate (0-1000 microM) all generated a concentration-dependent increase in carbonyl formation on BSA (107, 60, and 45%, respectively). GSNO did not affect carbonyl formation. Papain was inhibited by Angeli's salt, SIN-1, papanonoate, and GSNO with IC50 values of 0.62, 2.3, 54, and 80 microM, respectively. Angeli's salt (3.16 microM)-induced papain inactivation was only partially reversible, while the effects of GSNO (316 microM) and papanonoate (316 microM) were reversible upon addition of excess DTT. The Angeli's salt-mediated DTT-irreversible inhibition of papain was prevented by GSNO or papanonoate pretreatment, hypothetically through mixed disulfide formation or S-nitrosylation of the catalytically critical thiol group of papain. These results, for the first time, compare the generation of carbonyls in proteins by Angeli's salt, papanonoate, and SIN-1. Furthermore, these results suggest that S-nitrosothiols may have a novel function in protecting critical thiols from irreversible oxidative damage.

Advanced oxidation protein products — biological marker of oxidative stress = Zaawansowane produkty utleniania białek – biologiczne markery stresu oksydacyjnego

Directory of Open Access Journals (Sweden)

Anna Cwynar

2016-09-01

  ABSTRACT Advanced oxidation protein products (AOPPs are mostly derivatives of oxidatively modified albumin. The results of many experimental studies confirm intensification of oxidative modifications of proteins and an increase in concentration of advanced oxidation protein products (AOPPs in different pathological conditions, particularly those with well documented involvement of oxidative stress in their etiopathogenesis, but also those where its role is not yet well understood. Currently intensive research is carried out on the possibility of using AOPPs as useful indicators for diagnosing, prognosis and monitoring of diseases.   Keywords: advanced oxidation protein products, autoimmune disease, oxidative stress   STRESZCZENIE Zaawansowane produkty utleniania białek (AOPPs, to najczęściej pochodne zmodyfikowanej oksydacyjnie albuminy. Wyniki licznych badań doświadczalnych potwierdzają nasilenie oksydacyjnych modyfikacji białek i wzrost stężenia zaawansowanych produktów utleniania białek (AOPPs w różnych stanach patologicznych, szczególnie tych o dobrze udokumentowanym udziale stresu oksydacyjnego w ich etiopatogenezie, ale także takich, w których jego rola nie jest jeszcze dobrze poznana.. Obecnie trwają intensywne badania nad możliwością wykorzystania AOPPs, jako użytecznych wskaźników do diagnozowania, prognozowania oraz monitorowania chorób.   Słowa kluczowe: zaawansowane produkty utleniania białek, choroby autoimmunologiczne, stres oksydacyjny

Protein-bound tyrosine oxidation, nitration and chlorination by-products assessed by ultraperformance liquid chromatography coupled to tandem mass spectrometry.

Science.gov (United States)

Torres-Cuevas, Isabel; Kuligowski, Julia; Cárcel, María; Cháfer-Pericás, Consuelo; Asensi, Miguel; Solberg, Rønnaug; Cubells, Elena; Nuñez, Antonio; Saugstad, Ola Didrik; Vento, Máximo; Escobar, Javier

2016-03-24

Free radicals cause alterations in cellular protein structure and function. Oxidized, nitrated, and chlorinated modifications of aromatic amino acids including phenylalanine and tyrosine are reliable biomarkers of oxidative stress and inflammation in clinical conditions. To develop, validate and apply a rapid method for the quantification of known hallmarks of tyrosine oxidation, nitration and chlorination in plasma and tissue proteins providing a snapshot of the oxidative stress and inflammatory status of the organism and of target organs respectively. The extraction and clean up procedure entailed protein precipitation, followed by protein re-suspension and enzymatic digestion with pronase. An Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) method was developed to quantify protein released ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-nitrotyrosine (3NO2-Tyr) and 3-chlorotyrosine (3Cl-Tyr) as well as native phenylalanine (Phe) and tyrosine (p-Tyr) in plasma and tissue from a validated hypoxic newborn piglet experimental model. In plasma there was a significant increase in the 3NO2-Tyr/p-Tyr ratio. On the other hand m-Tyr/Phe and 3Cl-Tyr/p-Tyr ratios were significantly increased in liver of hypoxic compared with normoxic animals. Although no significant differences were found in brain tissue, a clear tendency to increased ratios was observed under hypoxic conditions. UPLC-MS/MS has proven suitable for the analysis of plasma and tissue samples from newborn piglets. The analysis of biomarkers of protein oxidation, nitration and chlorination will be applied in future studies aiming to provide a deeper insight into the mechanisms of oxidation-derived protein modification caused during neonatal asphyxia and resuscitation. Copyright © 2016 Elsevier B.V. All rights reserved.

Impact of acid and oxidative modifications, single or dual, of sorghum starch on biodegradable films.

Science.gov (United States)

Biduski, Bárbara; Silva, Francine Tavares da; Silva, Wyller Max da; Halal, Shanise Lisie de Mello El; Pinto, Vania Zanella; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

2017-01-01

The objective of this study was to evaluate the effects of acid and oxidation modifications on sorghum starch, as well as the effect of dual modification of starch on the physical, morphological, mechanical, and barrier properties of biodegradable films. The acid modification was performed with 3% lactic acid and the oxidation was performed with 1.5% active chlorine. For dual modification, the acid modification was performed first, followed by oxidation under the same conditions as above. Both films of the oxidized starches, single and dual, had increased stiffness, providing a higher tensile strength and lower elongation when compared to films based on native and single acid modified starches. However, the dual modification increased the water vapor permeability of the films without changing their solubility. The increase in sorghum starch concentration in the filmogenic solution increased the thickness, water vapor permeability, and elongation of the films. Copyright © 2016. Published by Elsevier Ltd.

Proteins oxidation and autoantibodies' reactivity against hydrogen peroxide and malondialdehyde -oxidized thyroid antigens in patients' plasmas with Graves' disease and Hashimoto Thyroiditis.

Science.gov (United States)

Mseddi, Malek; Ben Mansour, Riadh; Gargouri, Bochra; Mnif, Fatma; El Ghawi, Samir; Hammami, Boutheina; Ghorbel, Abdelmonem; Abid, Mohamed; Lassoued, Saloua

2017-06-25

The aim of this study was to evaluate proteins oxidation in plasmas of two autoimmune thyroid diseases (AITD): Graves' disease (GD) and Hashimoto Thyroiditis (HT), and to determine whether oxidative modification of thyroid antigens (T.Ag) enhanced the reactivity of autoantibodies in plasmas of AITD patients compared with the reactivity towards native T.Ag. Carbonyl and thiol groups and MDA-protein adducts were assessed spectrophotometric methods in plasmas of 74 AITD patients and 65 healthy controls. The reactivities immunoglobulin (Ig)G autoantibodies towards malondialdéhyde (MDA)-modified T.Ag, hydrogen peroxide (H 2 O 2 )-modified T.Ag, native T.Ag and native derm were checked by enzyme-linked immunosorbent assay (ELISA). Evaluation of oxidized proteins exhibited high levels of MDA bound to proteins and carbonyl groups, as well as reduced thiol level in plasmas of AITD patients by comparison to healthy controls (p thyroid stimulating hormone level in HT patients in the other (r = 0.65, p < 0.001). The data suggest that high production of H 2 O 2 probably occurred during hormone synthesis could contribute to protein oxidation in AITD and to create neoepitopes responsible for autoantibody reactivity's to H 2 O 2 -oxidized T.Ag enhancement. These results provide support to the involvement of oxidative stress in AITD development and/or exacerbation. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteomic Investigation of Protein Profile Changes and Amino Acid Residue Level Modification in Cooked Lamb Meat: The Effect of Boiling.

Science.gov (United States)

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2015-10-21

Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods.

Peptidomics of Peptic Digest of Selected Potato Tuber Proteins: Post-Translational Modifications and Limited Cleavage Specificity.

Science.gov (United States)

C K Rajendran, Subin R; Mason, Beth; Udenigwe, Chibuike C

2016-03-23

Bioinformatic tools are useful in predicting bioactive peptides from food proteins. This study was focused on using bioinformatics and peptidomics to evaluate the specificity of peptide release and post-translational modifications (PTMs) in a peptic digest of potato protein isolate. Peptides in the protein hydrolysate were identified by LC-MS/MS and subsequently aligned to their parent potato tuber proteins. Five major proteins were selected for further analysis, namely, lipoxygenase, α-1,4-glucan phosphorylase, annexin, patatin, and polyubiquitin, based on protein coverage, abundance, confidence levels, and function. Comparison of the in silico peptide profile generated with ExPASy PeptideCutter and experimental peptidomics data revealed several differences. The experimental peptic cleavage sites were found to vary in number and specificity from PeptideCutter predictions. Average peptide chain length was also found to be higher than predicted with hexapeptides as the smallest detected peptides. Moreover, PTMs, particularly Met oxidation and Glu/Asp deamidation, were observed in some peptides, and these were unaccounted for during in silico analysis. PTMs can be formed during aging of potato tubers, or as a result of processing conditions during protein isolation and hydrolysis. The findings provide insights on the limitations of current bioinformatics tools for predicting bioactive peptide release from proteins, and on the existence of structural modifications that can alter the peptide bioactivity and functionality.

Markers of protein oxidation

DEFF Research Database (Denmark)

Headlam, Henrietta A; Davies, Michael Jonathan

2004-01-01

Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed...... of this process depends on the extent of oxidation at C-3 compared with other sites. HO*, generated by gamma radiolysis, gave the highest total carbonyl yield, with protein-bound carbonyls predominating over released. In contrast, metal ion/H2O2 systems, gave more released than bound carbonyls, with this ratio...... modulated by EDTA. This is ascribed to metal ion-protein interactions affecting the sites of initial oxidation. Hypochlorous acid gave low concentrations of released carbonyls, but high yields of protein-bound material. The peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride...

Artificial Metalloenzymes through Chemical Modification of Engineered Host Proteins

KAUST Repository

Zernickel, Anna

2014-10-01

With a few exceptions, all organisms are restricted to the 20 canonical amino acids for ribosomal protein biosynthesis. Addition of new amino acids to the genetic code can introduce novel functionalities to proteins, broadening the diversity of biochemical as well as chemical reactions and providing new tools to study protein structure, reactivity, dynamics and protein-protein-interactions. The site directed in vivo incorporation developed by P. G. SCHULTZ and coworkers, using an archeal orthogonal tRNA/aaRS (aminoacyl-tRNA synthase) pair, allows site-specifically insertion of a synthetic unnatural amino acid (UAA) by reprogramming the amber TAG stop codon. A variety of over 80 different UAAs can be introduced by this technique. However by now a very limited number can form kinetically stable bonds to late transition metals. This thesis aims to develop new catalytically active unnatural amino acids or strategies for a posttranslational modification of site-specific amino acids in order to achieve highly enantioselective metallorganic enzyme hybrids (MOEH). As a requirement a stable protein host has to be established, surviving the conditions for incorporation, posttranslational modification and the final catalytic reactions. mTFP* a fluorescent protein was genetically modified by excluding any exposed Cys, His and Met forming a variant mTFP*, which fulfills the required specifications. Posttranslational chemical modification of mTFP* allow the introduction of single site metal chelating moieties. For modification on exposed cysteines different maleiimid containing ligand structures were synthesized. In order to perform copper catalyzed click reactions, suitable unnatural amino acids (para-azido-(L)-phenylalanine, para-ethynyl-(L)-phenylalanine) were synthesized and a non-cytotoxic protocol was established. The triazole ring formed during this reaction may contribute as a moderate σ-donor/π-acceptor ligand to the metal binding site. Since the cell limits the

PLMD: An updated data resource of protein lysine modifications.

Science.gov (United States)

Xu, Haodong; Zhou, Jiaqi; Lin, Shaofeng; Deng, Wankun; Zhang, Ying; Xue, Yu

2017-05-20

Post-translational modifications (PTMs) occurring at protein lysine residues, or protein lysine modifications (PLMs), play critical roles in regulating biological processes. Due to the explosive expansion of the amount of PLM substrates and the discovery of novel PLM types, here we greatly updated our previous studies, and presented a much more integrative resource of protein lysine modification database (PLMD). In PLMD, we totally collected and integrated 284,780 modification events in 53,501 proteins across 176 eukaryotes and prokaryotes for up to 20 types of PLMs, including ubiquitination, acetylation, sumoylation, methylation, succinylation, malonylation, glutarylation, glycation, formylation, hydroxylation, butyrylation, propionylation, crotonylation, pupylation, neddylation, 2-hydroxyisobutyrylation, phosphoglycerylation, carboxylation, lipoylation and biotinylation. Using the data set, a motif-based analysis was performed for each PLM type, and the results demonstrated that different PLM types preferentially recognize distinct sequence motifs for the modifications. Moreover, various PLMs synergistically orchestrate specific cellular biological processes by mutual crosstalks with each other, and we totally found 65,297 PLM events involved in 90 types of PLM co-occurrences on the same lysine residues. Finally, various options were provided for accessing the data, while original references and other annotations were also present for each PLM substrate. Taken together, we anticipated the PLMD database can serve as a useful resource for further researches of PLMs. PLMD 3.0 was implemented in PHP + MySQL and freely available at http://plmd.biocuckoo.org. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

2015-03-13

As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

International Nuclear Information System (INIS)

Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi; Zhang, Qunye; Li, Guorong

2015-01-01

As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation

Oxidatively Modified Proteins in the Serous Subtype of Ovarian Carcinoma

Directory of Open Access Journals (Sweden)

Sharifeh Mehrabi

2014-01-01

Full Text Available Serous subtype of ovarian cancer is considered to originate from fallopian epithelium mucosa that has been exposed to physiological changes resulting from ovulation. Ovulation influences an increased in inflammation of epithelial ovarian cells as results of constant exposure of cells to ROS. The imbalance between ROS and antioxidant capacities, as well as a disruption of redox signaling, causes a wide range of damage to DNA, proteins, and lipids. This study applied spectrophotometric, dinitrophenylhydrazone (DNPH assay, two-dimensional gel electrophoresis, and Western blot analyses to assess the levels of oxidatively modified proteins in 100 primary serous epithelial ovarian carcinoma and normal/surrounding tissues. These samples were obtained from 56 Caucasian and 44 African-American patients within the age range of 61±10 years. Analyses showed that the levels of reactive protein carbonyl groups increased as stages progressed to malignancy. Additionally, the levels of protein carbonyls in serous ovarian carcinoma among African Americans are 40% (P<0.05 higher relative to Caucasian at similar advanced stages. Results suggest that oxidative stress is involved in the modification of carbonyl protein groups, leading to increased aggressiveness of epithelial ovarian tumors and may contribute to the disease's invasiveness among African Americans.

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.

Science.gov (United States)

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

Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach

Energy Technology Data Exchange (ETDEWEB)

Dykstra, Andrew B [ORNL; Rodriguez, Jr., Miguel [ORNL; Raman, Babu [Dow Chemical Company, The; Cook, Kelsey [ORNL; Hettich, Robert {Bob} L [ORNL

2013-01-01

Post-translational modifications (PTMs) are known to play a significant role in many biological functions. The focus of this study is to characterize the post-translational modifications of the cellulosome protein complex used by the bacterium Clostridium thermocellum to better understand how this protein machine is tuned for enzymatic cellulose solubilization. To enhance comprehensive characterization, the extracellular cellulosome proteins were analyzed using multiple proteolytic digests (trypsin, Lys-C, Glu-C) and multiple fragmentation techniques (collisionally-activated dissociation, electron transfer dissociation, decision tree). As expected, peptide and protein identifications were increased by utilizing alternate proteases and fragmentation methods, in addition to the increase in protein sequence coverage. The complementarity of these experiments also allowed for a global exploration of PTMs associated with the cellulosome based upon a set of defined PTMs that included methylation, oxidation, acetylation, phosphorylation, and signal peptide cleavage. In these experiments, 85 modified peptides corresponding to 28 cellulosome proteins were identified. Many of these modifications were located in active cellulolytic or structural domains of the cellulosome proteins, suggesting a level of possible regulatory control of protein function in various cellulotyic conditions. The use of multiple enzymes and fragmentation technologies allowed for independent verification of PTMs in different experiments, thus leading to increased confidence in PTM identifications.

Impact of short-term dietary modification on postprandial oxidative stress

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Bloomer Richard J

2012-03-01

Full Text Available Abstract Background We have recently reported that short-term (21-day dietary modification in accordance with a stringent vegan diet (i.e., a Daniel Fast lowers blood lipids as well as biomarkers of oxidative stress. However, this work only involved measurements obtained in a fasted state. In the present study, we determined the postprandial response to a high-fat milkshake with regards to blood triglycerides (TAG, biomarkers of oxidative stress, and hemodynamic variables before and following a 21-day Daniel Fast. Methods Twenty-two subjects (10 men and 12 women; aged 35 ± 3 years completed a 21-day Daniel Fast. To induce oxidative stress, a milkshake (fat = 0.8 g·kg-1; carbohydrate = 1.0 g·kg-1; protein = 0.25 g·kg-1 was consumed by subjects on day one and day 22 in a rested and 12-hour fasted state. Before and at 2 and 4 h after consumption of the milkshake, heart rate (HR and blood pressure were measured. Blood samples were also collected at these times and analyzed for TAG, malondialdehyde (MDA, hydrogen peroxide (H2O2, advanced oxidation protein products (AOPP, nitrate/nitrite (NOx, and Trolox Equivalent Antioxidant Capacity (TEAC. Results A time effect was noted for HR (p = 0.006, with values higher at 2 hr post intake of the milkshake as compared to pre intake (p p = 0.02, and a trend for lower systolic blood pressure was noted (p = 0.07. Time effects were noted for TAG (p = 0.001, MDA (p 2O2 (p p p p p = 0.02, which was higher post fast as compared to pre fast. No pre/post fast × time interactions were noted (p > 0.05, with the area under the curve from pre to post fast reduced only slightly for TAG (11%, MDA (11%, H2O2 (8%, and AOPP (12%, with a 37% increase noted for NOx. Conclusion Partaking in a 21-day Daniel Fast does not result in a statistically significant reduction in postprandial oxidative stress. It is possible that a longer time course of adherence to the Daniel Fast eating plan may be needed to observe significant

Redox modification of caveolar proteins in the cardiovascular system- role in cellular signalling and disease.

Science.gov (United States)

Bubb, Kristen J; Birgisdottir, Asa Birna; Tang, Owen; Hansen, Thomas; Figtree, Gemma A

2017-08-01

Rapid and coordinated release of a variety of reactive oxygen species (ROS) such as superoxide (O 2 .- ), hydrogen peroxide (H 2 O 2 ) and peroxynitrite, in specific microdomains, play a crucial role in cell signalling in the cardiovascular system. These reactions are mediated by reversible and functional modifications of a wide variety of key proteins. Dysregulation of this oxidative signalling occurs in almost all forms of cardiovascular disease (CVD), including at the very early phases. Despite the heavily publicized failure of "antioxidants" to improve CVD progression, pharmacotherapies such as those targeting the renin-angiotensin system, or statins, exert at least part of their large clinical benefit via modulating cellular redox signalling. Over 250 proteins, including receptors, ion channels and pumps, and signalling proteins are found in the caveolae. An increasing proportion of these are being recognized as redox regulated-proteins, that reside in the immediate vicinity of the two major cellular sources of ROS, nicotinamide adenine dinucleotide phosphate oxidase (Nox) and uncoupled endothelial nitric oxide synthase (eNOS). This review focuses on what is known about redox signalling within the caveolae, as well as endogenous protective mechanisms utilized by the cell, and new approaches to targeting dysregulated redox signalling in the caveolae as a therapeutic strategy in CVD. Copyright © 2017. Published by Elsevier Inc.

Oxidation and nitrosation of meat proteins under gastro-intestinal conditions: Consequences in terms of nutritional and health values of meat.

Science.gov (United States)

de La Pomélie, Diane; Santé-Lhoutellier, Véronique; Sayd, Thierry; Gatellier, Philippe

2018-03-15

The chemical changes (oxidation/nitrosation) of meat proteins during digestion lead to a decrease in their nutritional value. Moreover, oxidized and nitrosated amino acids are suspected to promote various human pathologies. To investigate the mechanisms and the kinetics of these endogenous protein modifications, we used a dynamic artificial digestive system (DIDGI®) that mimics the physicochemical conditions of digestion. The combined effect of meat cooking and endogenous addition of ascorbate and nitrite was evaluated on protein oxidation (by measuring carbonyl groups), protein nitrosation (by measuring nitrosamines), and proteolysis. Considerable carbonylation was observed in the digestive tract, especially under the acidic conditions of the stomach. Nitrosamines, caused by ammonia oxidation, were formed in conditions in which no nitrite was added, although the addition of nitrite in the model significantly increased their levels. Meat cooking and nitrite addition significantly decreased protein digestion. The interactions between all the changes affecting the proteins are discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sequence tagging reveals unexpected modifications in toxicoproteomics

Science.gov (United States)

Dasari, Surendra; Chambers, Matthew C.; Codreanu, Simona G.; Liebler, Daniel C.; Collins, Ben C.; Pennington, Stephen R.; Gallagher, William M.; Tabb, David L.

2010-01-01

Toxicoproteomic samples are rich in posttranslational modifications (PTMs) of proteins. Identifying these modifications via standard database searching can incur significant performance penalties. Here we describe the latest developments in TagRecon, an algorithm that leverages inferred sequence tags to identify modified peptides in toxicoproteomic data sets. TagRecon identifies known modifications more effectively than the MyriMatch database search engine. TagRecon outperformed state of the art software in recognizing unanticipated modifications from LTQ, Orbitrap, and QTOF data sets. We developed user-friendly software for detecting persistent mass shifts from samples. We follow a three-step strategy for detecting unanticipated PTMs in samples. First, we identify the proteins present in the sample with a standard database search. Next, identified proteins are interrogated for unexpected PTMs with a sequence tag-based search. Finally, additional evidence is gathered for the detected mass shifts with a refinement search. Application of this technology on toxicoproteomic data sets revealed unintended cross-reactions between proteins and sample processing reagents. Twenty five proteins in rat liver showed signs of oxidative stress when exposed to potentially toxic drugs. These results demonstrate the value of mining toxicoproteomic data sets for modifications. PMID:21214251

Hypoxia-induced oxidative base modifications in the VEGF hypoxia-response element are associated with transcriptionally active nucleosomes.

Science.gov (United States)

Ruchko, Mykhaylo V; Gorodnya, Olena M; Pastukh, Viktor M; Swiger, Brad M; Middleton, Natavia S; Wilson, Glenn L; Gillespie, Mark N

2009-02-01

Reactive oxygen species (ROS) generated in hypoxic pulmonary artery endothelial cells cause transient oxidative base modifications in the hypoxia-response element (HRE) of the VEGF gene that bear a conspicuous relationship to induction of VEGF mRNA expression (K.A. Ziel et al., FASEB J. 19, 387-394, 2005). If such base modifications are indeed linked to transcriptional regulation, then they should be detected in HRE sequences associated with transcriptionally active nucleosomes. Southern blot analysis of the VEGF HRE associated with nucleosome fractions prepared by micrococcal nuclease digestion indicated that hypoxia redistributed some HRE sequences from multinucleosomes to transcriptionally active mono- and dinucleosome fractions. A simple PCR method revealed that VEGF HRE sequences harboring oxidative base modifications were found exclusively in mononucleosomes. Inhibition of hypoxia-induced ROS generation with myxathiozol prevented formation of oxidative base modifications but not the redistribution of HRE sequences into mono- and dinucleosome fractions. The histone deacetylase inhibitor trichostatin A caused retention of HRE sequences in compacted nucleosome fractions and prevented formation of oxidative base modifications. These findings suggest that the hypoxia-induced oxidant stress directed at the VEGF HRE requires the sequence to be repositioned into mononucleosomes and support the prospect that oxidative modifications in this sequence are an important step in transcriptional activation.

Radiation-induced reductive modifications of sulfur-containing amino acids within peptides and proteins.

Science.gov (United States)

Chatgilialoglu, Chryssostomos; Ferreri, Carla; Torreggiani, Armida; Salzano, Anna Maria; Renzone, Giovanni; Scaloni, Andrea

2011-10-19

The complex scenario of radical stress reactions affecting peptides/proteins can be better elucidated through the design of biomimetic studies simulating the consequences of the different free radicals attacking amino acids. In this context, ionizing radiations allowed to examine the specific damages caused by H-atoms and electrons coupled with protons, thus establishing the molecular basis of reductive radical stress. This is an innovative concept that complements the well-known oxidative stress also in view of a complete understanding of the global consequences of radical species reactivities on living systems. This review summarizes the knowledge of the chemical changes present in sulfur-containing amino acids occurring in polypeptides under reductive radical conditions, in particular the transformation of Met and Cys residues into α-amino butyric acid and alanine, respectively. Reductive radical stress causing a desulfurization process, is therefore coupled with the formation of S-centered radicals, which in turn can diffuse apart and become responsible of the damage transfer from proteins to lipids. These reductive modifications assayed in different peptide/protein sequences constitute an integration of the molecular inventories that up to now take into account only oxidative transformations. They can be useful to achieve an integrated vision of the free radical reactivities in a multifunctional system and, overall, for wider applications in the redox proteomics field. Copyright © 2011 Elsevier B.V. All rights reserved.

Immuno-Spin Trapping-Based Detection of Oxidative Modifications in Cardiomyocytes and Coronary Endothelium in the Progression of Heart Failure in Tgαq*44 Mice

Directory of Open Access Journals (Sweden)

Bartosz Proniewski

2018-05-01

Full Text Available Recent studies suggest both beneficial and detrimental role of increased reactive oxygen species and oxidative stress in heart failure (HF. However, it is not clear at which stage oxidative stress and oxidative modifications occur in the endothelium in relation to cardiomyocytes in non-ischemic HF. Furthermore, most methods used to date to study oxidative stress are either non-specific or require tissue homogenization. In this study, we used immuno-spin trapping (IST technique with fluorescent microscopy-based detection of DMPO nitrone adducts to localize and quantify oxidative modifications of the hearts from Tgαq*44 mice; a murine model of HF driven by cardiomyocyte-specific overexpression of Gαq* protein. Tgαq*44 mice and age-matched FVB controls at early, transition, and late stages of HF progression were injected with DMPO in vivo and analyzed ex vivo for DMPO nitrone adducts signals. Progressive oxidative modifications in cardiomyocytes, as evidenced by the elevation of DMPO nitrone adducts, were detected in hearts from 10- to 16-month-old, but not in 8-month-old Tgαq*44 mice, as compared with age-matched FVB mice. The DMPO nitrone adducts were detected in left and right ventricle, septum, and papillary muscle. Surprisingly, significant elevation of DMPO nitrone adducts was also present in the coronary endothelium both in large arteries and in microcirculation simultaneously, as in cardiomyocytes, starting from 10-month-old Tgαq*44 mice. On the other hand, superoxide production in heart homogenates was elevated already in 6-month-old Tgαq*44 mice and progressively increased to high levels in 14-month-old Tgαq*44 mice, while the enzymatic activity of catalase, glutathione reductase, and glutathione peroxidase was all elevated as early as in 4-month-old Tgαq*44 mice and stayed at a similar level in 14-month-old Tgαq*44. In summary, this study demonstrates that IST represents a unique method that allows to quantify oxidative

Myeloperoxidase-mediated protein lysine oxidation generates 2-aminoadipic acid and lysine nitrile in vivo.

Science.gov (United States)

Lin, Hongqiao; Levison, Bruce S; Buffa, Jennifer A; Huang, Ying; Fu, Xiaoming; Wang, Zeneng; Gogonea, Valentin; DiDonato, Joseph A; Hazen, Stanley L

2017-03-01

Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2-AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or base-catalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate protein-bound 2-AAA from an alternative potential lysine oxidative product, lysine nitrile (LysCN). Using a validated protease-based digestion method coupled with stable isotope dilution LC/MS/MS, we now report protein bound 2-AAA and LysCN are both formed by hypochlorous acid (HOCl) and the MPO/H 2 O 2 /Cl - system of leukocytes. At low molar ratio of oxidant to target protein N ε -lysine moiety, 2-AAA is formed via an initial N ε -monochloramine intermediate, which ultimately produces the more stable 2-AAA end-product via sequential generation of transient imine and semialdehyde intermediates. At higher oxidant to target protein N ε -lysine amine ratios, protein-bound LysCN is formed via initial generation of a lysine N ε -dichloramine intermediate. In studies employing MPO knockout mice and an acute inflammation model, we show that both free and protein-bound 2-AAA, and in lower yield, protein-bound LysCN, are formed by MPO in vivo during inflammation. Finally, both 2-AAA and to lesser extent LysCN are shown to be enriched in human aortic atherosclerotic plaque, a tissue known to harbor multiple MPO-catalyzed protein oxidation products. Collectively, these results show that MPO-mediated oxidation of protein lysyl

Chaperones, but not oxidized proteins, are ubiquitinated after oxidative stress

DEFF Research Database (Denmark)

Kästle, Marc; Reeg, Sandra; Rogowska-Wrzesinska, Adelina

2012-01-01

of these proteins by MALDI tandem mass spectrometry (MALDI MS/MS). As a result we obtained 24 different proteins which can be categorized into the following groups: chaperones, energy metabolism, cytoskeleton/intermediate filaments, and protein translation/ribosome biogenesis. The special set of identified......, ubiquitinated proteins confirm the thesis that ubiquitination upon oxidative stress is no random process to degrade the mass of oxidized proteins, but concerns a special group of functional proteins....

Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins

OpenAIRE

Deming, TJ

2017-01-01

Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-cont...

Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins.

Science.gov (United States)

Deming, Timothy J

2017-03-15

Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-containing amino acids has many advantages and is a complementary methodology to the widely utilized methods for modification at cysteine residues.

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

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M. Ryan Smith

2016-08-01

Full Text Available Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP, decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231 breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC protein levels, although other protein levels were

Ferulic acid modification enhances the anti-oxidation activity of natural Hb in vitro.

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Qi, Donglai; Li, Qian; Chen, Chen; Wang, Xiang

2018-03-13

During the development of artificial red blood cell (RBC) substitutes, oxidation side reaction is one of the major factors that hinder the application of haemoglobin (Hb)-based oxygen carriers (HBOCs). In order to avoid oxidation toxicity, we designed and prepared natural Hb conjugated with ferulic acid (FA) via simple chemical modification. In addition, the thiol groups on Hb surface were increased via the reaction of Hb with 2-iminothiolane (2-IT) and then modified with FA for the study of anti-oxidant ability. It was showed that Hb modified with FA (FA-Hb) had similar oxygen-binding capacity to natural Hb. Moreover, the anti-oxidant ability of FA-Hb in vitro in different systems was superior to natural Hb and in proportion to the degree of modification of FA. The results indicate that FA-Hb might have the potential to serve as a novel oxygen carrier with the capacity to reduce oxidative side reaction.

Protein capped nanosilver free radical oxidation: role of biomolecule capping on nanoparticle colloidal stability and protein oxidation.

Science.gov (United States)

Ahumada, Manuel; Bohne, Cornelia; Oake, Jessy; Alarcon, Emilio I

2018-05-03

We studied the effect of human serum albumin protein capped spherical nanosilver on the nanoparticle stability upon peroxyl radical oxidation. The nanoparticle-protein composite is less prone to oxidation compared to the individual components. However, higher concentrations of hydrogen peroxide were formed in the nanoparticle-protein system.

Cell signaling, post-translational protein modifications and NMR spectroscopy

International Nuclear Information System (INIS)

Theillet, Francois-Xavier; Smet-Nocca, Caroline; Liokatis, Stamatios; Thongwichian, Rossukon; Kosten, Jonas; Yoon, Mi-Kyung; Kriwacki, Richard W.; Landrieu, Isabelle; Lippens, Guy; Selenko, Philipp

2012-01-01

Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.

Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials

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Bloom, Steven; Liu, Chun; Kölmel, Dominik K.; Qiao, Jennifer X.; Zhang, Yong; Poss, Michael A.; Ewing, William R.; MacMillan, David W. C.

2018-02-01

The advent of antibody-drug conjugates as pharmaceuticals has fuelled a need for reliable methods of site-selective protein modification that furnish homogeneous adducts. Although bioorthogonal methods that use engineered amino acids often provide an elegant solution to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, we explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, we demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidation potentials between internal versus C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidation potential-gated technology is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodology provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation.

Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials.

Science.gov (United States)

Bloom, Steven; Liu, Chun; Kölmel, Dominik K; Qiao, Jennifer X; Zhang, Yong; Poss, Michael A; Ewing, William R; MacMillan, David W C

2018-02-01

The advent of antibody-drug conjugates as pharmaceuticals has fuelled a need for reliable methods of site-selective protein modification that furnish homogeneous adducts. Although bioorthogonal methods that use engineered amino acids often provide an elegant solution to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, we explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, we demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidation potentials between internal versus C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidation potential-gated technology is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodology provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation.

Posttranslational modification of bioaerosol protein by common gas pollutants: NO2 and O3

Science.gov (United States)

Abdullahi Mahmood, Marliyyah; Bloss, William; Pope, Francis

2016-04-01

Air pollution can exacerbate several medical conditions, for example, hay fever and asthma. The global incidence of hay fever has been rising for decades; however, the underlying reasons behind this rise remain unclear. It is hypothesized that the exposure of pollen to common gas phase pollutants, such as nitrogen dioxide (NO2) and ozone (O3), increases the allergenicity of the pollen and thus increases hay fever incidence (Reinmuth-Selzle et al., 2014, Franze, et al., 2005). Since atmospheric pollutants often have greater concentrations within urban areas (in particular NO2) the hypothesis suggests that greater allergenicity should occur in urban areas. Certainly, several studies do suggest higher hay fever incidence within urban areas compared to rural areas (Schröder et al., 2015). Previous published work suggests a link between increased allergies and changes in the chemical composition of pollen protein via posttranslational modification of the protein (Reinmuth-Selzle et al., 2014). This study investigates the posttranslational modification of two highly allergenic pollen species (Birch and Ragweed) that are common in Europe. Within the laboratory, we expose pollen grains to atmospherically relevant exposures of gas phase NO2, O3 and other common gas phase oxidants under a range of environmentally relevant conditions. The effects of the exposures on the biochemistry of the pollen grains were probed using a proteomic approach (liquid chromatography coupled ultra-high resolution spectrometer). Our findings indicate the interaction between gas phase pollutants and pollen cause protein specific modifications; in particular nitration that occurs upon tyrosine residues and nitrosylation on cysteine residues. These modifications may affect human immune response to the pollen protein, which may suggest a possible reason for increased allergies in reaction to such chemically altered protein. Quantification of the relative degree of PTMs, from a variety of

Peculiarities of oxidative modification of proteins indices in blood plasma of the female rats’ offspring with experimental gestational diabetes depending on sex, age and basal glycemia level

Directory of Open Access Journals (Sweden)

O. V. Gancheva

2015-04-01

Full Text Available Analysis of products of oxidative modification of proteins (OMP in correlation with plasma catalase activity will help us to define the state of oxidative stress parameters in the normal animals and in the female rats’ offspring with experimental gestational diabetes (EGD. Itwilldefine their role in organism’s reorganization aimed to activation of compensatory and defensive mechanisms both at the cellular level and at the level of homeostasis. We’ll also define its alteration in animals with the prenatal negative influence of chronic hyperglycemia. The aim of research wasto study peculiarities of OMP parameters and plasma catalase activity of the female rats’offspring with EGD in dependence on sex, age and basal glycemia level. Materials and methods.The research was carried out on 80 rats (males and females offspring of the female rats with normal pregnancy and 80 offspring (males and females of the female rats with EGD. The animals were grouped by the age 2, 4, 6 and 18 months; 20 animals in each group. Animals were freely allowed to standard food and water. When animals reached appropriate age they were decapitated under thiopental anesthesia (40 mg/kg. Glucose was measured by glucose oxidative method in all groups of animals. In order to define the intensity of oxidative reactions in blood plasma of laboratory animals the degree of OMP by Halliwell method was done. Wemeasuredlevelofaldehydephenylhydrazone (APH which is thought to be as early mark of proteins oxidation, and ketonephenylhydrazone (KPH which is referred to late mark of protein destruction. The state of antioxidative system was evaluated by plasma catalase activity with the help of spectrophotometric method. The obtained data was processed by statistic programs VIDAS-2.5 (Kontron Elektronik, Германия, EXCEL MS Office 2007 (Microsoft Corp., США, STATISTICA 6.0 (Stat-Soft, 2001. Results and discussion. In the present research, we have observed the decrease of

Determination of plasminogen/plasmin system components and indicators of lipoproteins oxidative modification under arterial hypertension

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O. I. Yusova

2018-02-01

Full Text Available The present study was investigated of levels of oxidative modification of lipoproteins and content of plasminogen/plasmin system components – tissue-type plasminogen activator (t-PA and plasminogen activators inhibitor-1 (PAI-I, in patients with stage II arterial hypertension (AHT and resistant form. It was established that t-PA level in blood plasma of the patients is 2 times lower under stage II hypertension than normal and 2.5 times lower under resistant AHT. The inhibitor activity is 1.5 and 2 times higher consequently. It is concluded that patients with AHT have a decreased fibrinolytic potential, which can cause thrombotic states. Our evaluation showed a significant accumulation of products of lipid and protein oxidation, decrease of activity of antioxidant enzymes and changes of the activity of high density-lipoproteins-associated enzymes (decrease of paraoxonase-1 activity, increase of myeloperoxidase activity. Oxidized lipoproteins, t-PA and PAI-1 can be used as prognostic markers of development of complications and for evaluating the efficacy of therapy in patients with arterial hypertension.

Spatial and Temporal Effects in Protein Post-translational Modification Distributions in the Developing Mouse Brain

DEFF Research Database (Denmark)

Edwards, Alistair V G; Edwards, Gregory J; Schwämmle, Veit

2014-01-01

Protein post-translational modification (PTM) is a powerful way to modify the behavior of cellular proteins and thereby cellular behavior. Multiple recent studies of evolutionary trends have shown that certain pairs of protein post-translational modifications tend to occur closer to each other than...... for observations of increasingly frequent and diverse protein modification in cell biology. In this study, we use mass spectrometry and proteomic strategies to present biological data showing spatiotemporal PTM co-localization across multiple PTM categories, which display changes over development of the brain...

Protein modification by acrolein: Formation and stability of cysteine adducts

OpenAIRE

Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

2009-01-01

The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to iden...

Pattern of occurrence and occupancy of carbonylation sites in proteins

DEFF Research Database (Denmark)

Rao, R Shyama Prasad; Møller, Ian Max

2011-01-01

sites. Comparison of metal-catalyzed oxidation of two closely related proteins indicates that this type of carbonylation might not be very specific in proteins. Interestingly, carbonylated sites show a very strong tendency to cluster together in the protein primary sequence hinting at some sort......Proteins are targets for modification by reactive oxygen species, and carbonylation is an important irreversible modification that increases during oxidative stress. While information on protein carbonylation is accumulating, its pattern is not yet understood. We have made a meta...

Capture of negative muons in magnesium oxides and crystalline modifications of phosphorus

International Nuclear Information System (INIS)

Zinov, V.G.; Kachalkin, A.K.; Nikityuk, L.N.; Pokrovskij, V.N.; Rybakov, V.N.; Yutlandov, I.A.

1977-01-01

The paper is aimed at comparing the structure of mesic K X-ray patterns of phosphorus in its crystalline modifications, comparing the structure of mesic X-ray patterns of magnesium and oxygen in compounds of MgO, MgO 2 , H 2 O and metallic magnesium, as well as comparison of propabilities of μ - atomic capture in magnesium oxides. By analyzing the mesic K X-ray patterns of red and white phosphorus it is concluded that the phosphorus crystalline modification produces the effect on the line structure, the higher series number being somewhat larger for the allotrope of phosphorus with polymeric structure. A comparison is made of the mesic X-ray series of the magnesium in oxide and metal, of the oxygen in oxide and water with the analogous data for aluminium and silicon. The data confirm the supposition that chemical bond (valence electrons) plays a substantial role in meson capture

Lipids and Protein Peroxidation in Children and Teenager Patients with Pulmonary Tuberculosis

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Yu.V. Poliakova

2015-09-01

Full Text Available A review of literature about the study of lipid and protein peroxidation in children and teenagers with pulmonary tuberculosis nowadays was carried out. It was established that there is a great number works dedicated to the lipid peroxidation and antioxidant protective system in various pathological conditions of the respiratory system, including pulmonary tuberculosis in children and teenagers today. Oxidative modification proteins products are the earliest markers of oxidative stress in patients. There is no information on the oxidative modification of proteins in children and teenagers suffering from pulmonary tuberculosis in the literature. The study of oxidative modification of proteins will facilitate the development of more efficient new diagnosis methods and pathogenetic treatment of children and teenagers with pulmonary tuberculosis, that will increase the treatment effectiveness.

Modifications of proteins by polyunsaturated fatty acid peroxidation products

DEFF Research Database (Denmark)

Refsgaard, Hanne; Tsai, Lin; Stadtman, Earl

2000-01-01

The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(lll)/O-2] depends on the degree of unsaturation of the fatty acid. The fatty acid......-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate fatty acids were oxidized in the presence...... in the formation of protein carbonyls, These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (alpha,beta-unsaturated aldehydes) with lysine residues...

Overview of xeroderma pigmentosum proteins architecture, mutations and post-translational modifications.

Science.gov (United States)

Feltes, Bruno César; Bonatto, Diego

2015-01-01

The xeroderma pigmentosum complementation group proteins (XPs), which include XPA through XPG, play a critical role in coordinating and promoting global genome and transcription-coupled nucleotide excision repair (GG-NER and TC-NER, respectively) pathways in eukaryotic cells. GG-NER and TC-NER are both required for the repair of bulky DNA lesions, such as those induced by UV radiation. Mutations in genes that encode XPs lead to the clinical condition xeroderma pigmentosum (XP). Although the roles of XPs in the GG-NER/TC-NER subpathways have been extensively studied, complete knowledge of their three-dimensional structure is only beginning to emerge. Hence, this review aims to summarize the current knowledge of mapped mutations and other structural information on XP proteins that influence their function and protein-protein interactions. We also review the possible post-translational modifications for each protein and the impact of these modifications on XP protein functions. Copyright © 2014 Elsevier B.V. All rights reserved.

Alteration of human serum albumin binding properties induced by modifications: A review

Science.gov (United States)

Maciążek-Jurczyk, Małgorzata; Szkudlarek, Agnieszka; Chudzik, Mariola; Pożycka, Jadwiga; Sułkowska, Anna

2018-01-01

Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

Altering protein surface charge with chemical modification modulates protein–gold nanoparticle aggregation

International Nuclear Information System (INIS)

Jamison, Jennifer A.; Bryant, Erika L.; Kadali, Shyam B.; Wong, Michael S.; Colvin, Vicki L.; Matthews, Kathleen S.; Calabretta, Michelle K.

2011-01-01

Gold nanoparticles (AuNP) can interact with a wide range of molecules including proteins. Whereas significant attention has focused on modifying the nanoparticle surface to regulate protein–AuNP assembly or influence the formation of the protein “corona,” modification of the protein surface as a mechanism to modulate protein–AuNP interaction has been less explored. Here, we examine this possibility utilizing three small globular proteins—lysozyme with high isoelectric point (pI) and established interactions with AuNP; α-lactalbumin with similar tertiary fold to lysozyme but low pI; and myoglobin with a different globular fold and an intermediate pI. We first chemically modified these proteins to alter their charged surface functionalities, and thereby shift protein pI, and then applied multiple methods to assess protein–AuNP assembly. At pH values lower than the anticipated pI of the modified protein, AuNP exposure elicits changes in the optical absorbance of the protein–NP solutions and other properties due to aggregate formation. Above the expected pI, however, protein–AuNP interaction is minimal, and both components remain isolated, presumably because both species are negatively charged. These data demonstrate that protein modification provides a powerful tool for modulating whether nanoparticle–protein interactions result in material aggregation. The results also underscore that naturally occurring protein modifications found in vivo may be critical in defining nanoparticle–protein corona compositions.

Albumin modification and fragmentation in renal disease.

Science.gov (United States)

Donadio, Carlo; Tognotti, Danika; Donadio, Elena

2012-02-18

Albumin is the most important antioxidant substance in plasma and performs many physiological functions. Furthermore, albumin is the major carrier of endogenous molecules and exogenous ligands. This paper reviews the importance of post-translational modifications of albumin and fragments thereof in patients with renal disease. First, current views and controversies on renal handling of proteins, mainly albumin, will be discussed. Post-translational modifications, namely the fragmentation of albumin found with proteomic techniques in nephrotic patients, diabetics, and ESRD patients will be presented and discussed. It is reasonable to hypothesize that proteolytic fragmentation of serum albumin is due to a higher susceptibility to proteases, induced by oxidative stress. The clinical relevance of the fragmentation of albumin has not yet been established. These modifications could affect some physiological functions of albumin and have a patho-physiological role in uremic syndrome. Proteomic analysis of serum allows the identification of over-expressed proteins and can detect post-translational modifications of serum proteins, hitherto hidden, using standard laboratory techniques. Copyright © 2011 Elsevier B.V. All rights reserved.

Proteins and their modifications in a medieval mummy

Czech Academy of Sciences Publication Activity Database

Mikšík, Ivan; Sedláková, Pavla; Pataridis, Statis; Bortolotti, F.; Gottardo, R.

2016-01-01

Roč. 25, č. 11 (2016), s. 2037-2044 ISSN 0961-8368 R&D Projects: GA ČR(CZ) GA15-01948S Institutional support: RVO:67985823 Keywords : mummy * collagen * protein modification * deamidation * carbamylation * carboxymethylation Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.523, year: 2016

Antioxidant systems are regulated by nitric oxide-mediated post-translational modifications (NO-PTMs

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Juan Carlos Begara-Morales

2016-02-01

Full Text Available Nitric oxide (NO is a biological messenger that orchestrates a plethora of plant functions, mainly through post-translational modifications (PTMs such as S-nitrosylation or tyrosine nitration. In plants, hundreds of proteins have been identified as potential targets of these NO-PTMs under physiological and stress conditions indicating the relevance of NO in plant-signaling mechanisms. Among these NO protein targets, there are different antioxidant enzymes involved in the control of reactive oxygen species (ROS, such as H2O2, which is also a signal molecule. This highlights the close relationship between ROS/NO signaling pathways. The major plant antioxidant enzymes, including catalase, superoxide dismutases (SODs peroxiredoxins (Prx and all the enzymatic components of the ascorbate-glutathione (Asa-GSH cycle, have been shown to be modulated to different degrees by NO-PTMs. This mini-review will update the recent knowledge concerning the interaction of NO with these antioxidant enzymes, with a special focus on the components of the Asa-GSH cycle and their physiological relevance.

Hypochlorous and peracetic acid induced oxidation of dairy proteins.

Science.gov (United States)

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.

Protein N-myristoylation in Escherichia coli: Reconstitution of a eukaryotic protein modification in bacteria

International Nuclear Information System (INIS)

Duronio, R.J.; Jackson-Machelski, E.; Heuckeroth, R.O.; Gordon, J.I.; Olins, P.O.; Devine, C.S.; Yonemoto, W.; Slice, L.W.; Taylor, S.S.

1990-01-01

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH 2 -terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. The authors have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH 2 -terminal 39 amino acids. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed

Milk whey protein modification by coffee-specific phenolics: effect on structural and functional properties.

Science.gov (United States)

Ali, Mostafa; Homann, Thomas; Khalil, Mahmoud; Kruse, Hans-Peter; Rawel, Harshadrai

2013-07-17

A suitable vehicle for integration of bioactive plant constituents is proposed. It involves modification of proteins using phenolics and applying these for protection of labile constituents. It dissects the noncovalent and covalent interactions of β-lactoglobulin with coffee-specific phenolics. Alkaline and polyphenol oxidase modulated covalent reactions were compared. Tryptic digestion combined with MALDI-TOF-MS provided tentative allocation of the modification type and site in the protein, and an in silico modeling of modified β-lactoglobulin is proposed. The modification delivers proteins with enhanced antioxidative properties. Changed structural properties and differences in solubility, surface hydrophobicity, and emulsification were observed. The polyphenol oxidase modulated reaction provides a modified β-lactoglobulin with a high antioxidative power, is thermally more stable, requires less energy to unfold, and, when emulsified with lutein esters, exhibits their higher stability against UV light. Thus, adaptation of this modification provides an innovative approach for functionalizing proteins and their uses in the food industry.

The role of proteins in damage induced by free radicals

International Nuclear Information System (INIS)

Gebicki, J.M.

1996-01-01

The initial consequence of oxidative stress in living organisms is chemical modification of cell components. Recently increasing attention in this area has been paid to the modification of proteins. A form of protein modification which has been studied in some detail only recently is peroxidation. In the last 8 years, we and our collaborators have shown that a range of isolated proteins acquire hydroperoxide groups when exposed to a range of biologically plausible oxidants. These include HO free radicals generated by radiation or in the Fenton reaction, peroxyl radicals, oxidants released by activated neutrophils, and peroxynitrite. In more complex systems, we also found protein peroxides in the apo B component of LDL treated with 20 μM Cu ++ , and in irradiated blood serum. These observations suggest that the formation of protein peroxides is a possible consequence of oxidative stress in vivo. A remarkable feature of the process of protein peroxidation is its high efficiency. This is most easily measured with proteins oxidized by radiation-generated free radicals. It was found that, for some proteins, peroxide yields reached 40% of the numbers of HO radicals generated. Thus in effect, almost half of these radicals can be converted to the much more long-lived protein peroxide groups. If they, in turn, have the capacity to damage other molecules, the major oxidative pathway in vivo may have the sequence: free radical ? protein peroxide ? another oxidized molecule. This hypothesis was tested by studying the ability of protein peroxides to react with selected molecules and the results are briefly discussed. Clearly, these effects are specific to individual proteins. More generally, amino acid and protein peroxides were found to be a potential source of a range of free radicals when reduced by Fe ++ . If this turns out to be a common phenomenon, protein peroxides may prove to be a major source of oxidative damage

The role of proteins in damage induced by free radicals

Energy Technology Data Exchange (ETDEWEB)

Gebicki, J.M. [Macquarie Univ., North Ryde, NSW (Australia). School of Biological Sciences

1996-12-31

The initial consequence of oxidative stress in living organisms is chemical modification of cell components. Recently increasing attention in this area has been paid to the modification of proteins. A form of protein modification which has been studied in some detail only recently is peroxidation. In the last 8 years, we and our collaborators have shown that a range of isolated proteins acquire hydroperoxide groups when exposed to a range of biologically plausible oxidants. These include HO free radicals generated by radiation or in the Fenton reaction, peroxyl radicals, oxidants released by activated neutrophils, and peroxynitrite. In more complex systems, we also found protein peroxides in the apo B component of LDL treated with 20 {mu}M Cu{sup ++}, and in irradiated blood serum. These observations suggest that the formation of protein peroxides is a possible consequence of oxidative stress in vivo. A remarkable feature of the process of protein peroxidation is its high efficiency. This is most easily measured with proteins oxidized by radiation-generated free radicals. It was found that, for some proteins, peroxide yields reached 40% of the numbers of HO radicals generated. Thus in effect, almost half of these radicals can be converted to the much more long-lived protein peroxide groups. If they, in turn, have the capacity to damage other molecules, the major oxidative pathway in vivo may have the sequence: free radical ? protein peroxide ? another oxidized molecule. This hypothesis was tested by studying the ability of protein peroxides to react with selected molecules and the results are briefly discussed. Clearly, these effects are specific to individual proteins. More generally, amino acid and protein peroxides were found to be a potential source of a range of free radicals when reduced by Fe{sup ++}. If this turns out to be a common phenomenon, protein peroxides may prove to be a major source of oxidative damage.

Oxidative modification and electrochemical inactivation of Escherichia coli upon cold atmospheric pressure plasma exposure.

Directory of Open Access Journals (Sweden)

Marlène Dezest

Full Text Available Cold atmospheric pressure plasmas (CAPPs are known to have bactericidal effects but the mechanism of their interaction with microorganisms remains poorly understood. In this study the bacteria Escherichia coli were used as a model and were exposed to CAPPs. Different gas compositions, helium with or without adjunctions of nitrogen or oxygen, were used. Our results indicated that CAPP induced bacterial death at decontamination levels depend on the duration, post-treatment storage and the gas mixture composition used for the treatment. The plasma containing O2 in the feeding gas was the most aggressive and showed faster bactericidal effects. Structural modifications of treated bacteria were observed, especially significant was membrane leakage and morphological changes. Oxidative stress caused by plasma treatment led to significant damage of E. coli. Biochemical analyses of bacterial macromolecules indicated massive intracellular protein oxidation. However, reactive oxygen and nitrogen species (RONS are not the only actors involved in E. coli's death, electrical field and charged particles could play a significant role especially for He-O2 CAPP.

Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

International Nuclear Information System (INIS)

Inadomi, Chiaki; Murata, Hiroaki; Ihara, Yoshito; Goto, Shinji; Urata, Yoshishige; Yodoi, Junji; Kondo, Takahito; Sumikawa, Koji

2012-01-01

Highlights: ► GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. ► NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. ► Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

Post-Translational Modifications of Desulfovibrio vulgaris Hildenborough Sulfate Reduction Pathway Proteins

Energy Technology Data Exchange (ETDEWEB)

Gaucher, S.P.; Redding, A.M.; Mukhopadhyay, A.; Keasling, J.D.; Singh, A.K.

2008-03-01

Recent developments in shotgun proteomics have enabled high-throughput studies of a variety of microorganisms at a proteome level and provide experimental validation for predicted open reading frames in the corresponding genome. More importantly, advances in mass spectrometric data analysis now allow mining of large proteomics data sets for the presence of post-translational modifications(PTMs). Although PTMs are a critical aspectof cellular activity, such information eludes cell-wide studies conducted at the transcript level. Here, we analyze several mass spectrometric data sets acquired using two-dimensional liquid chromatography tandem mass spectrometry, 2D-LC/MS/MS, for the sulfate reducing bacterium, Desulfovibrio vulgaris Hildenborough. Our searches of the raw spectra led us to discover several post-translationally modified peptides in D. vulgaris. Of these, several peptides containing a lysine with a +42 Da modification were found reproducibly across all data sets. Both acetylation and trimethylation have the same nominal +42 Da mass, and are therefore candidates for this modification. Several spectra were identified having markers for trimethylation, while one is consistent with an acetylation. Surprisingly, these modified peptides predominantly mapped to proteins involved in sulfate respiration. Other highly expressed proteins in D. vulgaris, such as enzymes involved in electron transport and other central metabolic processes, did not contain this modification. Decoy database searches were used to control for random spectrum/sequence matches. Additional validation for these modifications was provided by alternate workflows, for example, two-dimensional gel electrophoresis followed by mass spectrometry analysis of the dissimilatory sulfite reductase gamma-subunit(DsrC) protein. MS data for DsrC in this alternate workflow also contained the +42 Da modification at the same loci. Furthermore, the DsrC homologue in another sulfate reducing bacterium

Beyond gene expression: the impact of protein post-translational modifications in bacteria.

Science.gov (United States)

Cain, Joel A; Solis, Nestor; Cordwell, Stuart J

2014-01-31

The post-translational modification (PTM) of proteins plays a critical role in the regulation of a broad range of cellular processes in eukaryotes. Yet their role in governing similar systems in the conventionally presumed 'simpler' forms of life has been largely neglected and, until recently, was thought to occur only rarely, with some modifications assumed to be limited to higher organisms alone. Recent developments in mass spectrometry-based proteomics have provided an unparalleled power to enrich, identify and quantify peptides with PTMs. Additional modifications to biological molecules such as lipids and carbohydrates that are essential for bacterial pathophysiology have only recently been detected on proteins. Here we review bacterial protein PTMs, focusing on phosphorylation, acetylation, proteolytic degradation, methylation and lipidation and the roles they play in bacterial adaptation - thus highlighting the importance of proteomic techniques in a field that is only just in its infancy. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. © 2013 Elsevier B.V. All rights reserved.

Maillard reaction versus other nonenzymatic modifications in neurodegenerative processes.

Science.gov (United States)

Pamplona, Reinald; Ilieva, Ekaterina; Ayala, Victoria; Bellmunt, Maria Josep; Cacabelos, Daniel; Dalfo, Esther; Ferrer, Isidre; Portero-Otin, Manuel

2008-04-01

Nonenzymatic protein modifications are generated from direct oxidation of amino acid side chains and from reaction of the nucleophilic side chains of specific amino acids with reactive carbonyl species. These reactions give rise to specific markers that have been analyzed in different neurodegenerative diseases sharing protein aggregation, such as Alzheimer's disease, Pick's disease, Parkinson's disease, dementia with Lewy bodies, Creutzfeldt-Jakob disease, and amyotrophic lateral sclerosis. Collectively, available data demonstrate that oxidative stress homeostasis, mitochondrial function, and energy metabolism are key factors in determining the disease-specific pattern of protein molecular damage. In addition, these findings suggest the lack of a "gold marker of oxidative stress," and, consequently, they strengthen the need for a molecular dissection of the nonenzymatic reactions underlying neurodegenerative processes.

Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification.

Science.gov (United States)

Wible, Ryan S; Sutter, Thomas R

2017-03-20

The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

Br2 induced oxidative pore modification of a porous coordination network.

Science.gov (United States)

Ohtsu, Hiroyoshi; Kawano, Masaki

2016-01-14

Iodinated pores of a Zn-based coordination network were modified by Br2 oxidation to produce brominated pores in a polycrystalline-to-polycrystalline manner while maintaining the same network topology. Ab initio X-ray powder diffraction analysis and Raman spectroscopy revealed that the brominated pore can trap Br2 or I2 by strong σ/π-type interactions. A kinetic study in solution revealed that the pore modification by Br2 oxidation is much faster than the Br2 encapsulation process.

Posttranslational modifications of proteins : tools for functional proteomics [Methods in molecular biology, v. 194

National Research Council Canada - National Science Library

Kannicht, Christoph

2002-01-01

... single glycosylation sites in a protein. Additional powerful techniques facilitate the analysis of glycosylphosphatidylinositols, lipid modifications, protein phosphorylation and sulfation, protein methylation and acetylation, a-amidation...

Oxidative DNA base modifications as factors in carcinogenesis

International Nuclear Information System (INIS)

Olinski, R.; Jaruga, P.; Zastawny, T.H.

1998-01-01

Reactive oxygen species can cause extensive DNA modifications including modified bases. Some of the DNA base damage has been found to possess premutagenic properties. Therefore, if not repaired, it can contribute to carcinogenesis. We have found elevated amounts of modified bases in cancerous and precancerous tissues as compared with normal tissues. Most of the agents used in anticancer therapy are paradoxically responsible for induction of secondary malignancies and some of them may generate free radicals. The results of our experiments provide evidence that exposure of cancer patients to therapeutic doses of ionizing radiation and anticancer drugs cause base modifications in genomic DNA of lymphocytes. Some of these base damages could lead to mutagenesis in critical genes and ultimately to secondary cancers such as leukemias. This may point to an important role of oxidative base damage in cancer initiation. Alternatively, the increased level of the modified base products may contribute to genetic instability and metastatic potential of tumor cells. (author)

Protein and lipid oxidative damage and complex I content are lower in the brain of budgerigar and canaries than in mice. Relation to aging rate.

Science.gov (United States)

Pamplona, Reinald; Portero-Otín, Manuel; Sanz, Alberto; Ayala, Victoria; Vasileva, Ekaterina; Barja, Gustavo

2005-12-01

What are the mechanisms determining the rate of animal aging? Of the two major classes of endothermic animals, bird species are strikingly long-lived compared to mammals of similar body size and metabolic rate. Thus, they are ideal models to identify longevity-related characteristics not linked to body size or low metabolic rates. Since oxidative stress seems to be related to the basic aging process, we measured specific markers of different kinds of oxidative damage to proteins, like glutamic and aminoadipic semialdehydes (GSA and AASA, specific protein carbonyls), Nɛ-(carboxyethyl)lysine (CEL), Nɛ-(carboxymethyl)lysine (CML), and Nɛ-(malondialdehyde)lysine (MDAL), as well as mitochondrial Complex I content and amino acid and membrane fatty acyl composition, in the brain of short-lived mice (maximum life span [MLSP] 3.5 years) compared with those of long-lived budgerigar 'parakeets' (MLSP, 21 years) and canaries (MLSP, 24 years). The brains of both bird species had significantly lower levels of compounds formed as a result of oxidative (GSA and AASA), glycoxidative (CEL and CML), and lipoxidative (CML and MDAL) protein modifications, as well as a lower levels of mitochondrial complex I protein. Although it is known that fatty acid unsaturation is lower in many tissues of long-lived compared to short-lived mammals, this is not true in the particular case of brain. In agreement with this, we also found that the brain tissue of bugerigars and canaries contains no fewer double bonds than that of mice. Amino acid composition analyses revealed that bird proteins have a significantly lower content of His, Leu and Phe, as well as, interestingly, of methionine, whereas Asp, Glu, Ala, Val, and Lys contents were higher than in the mammals. These results, together with those previously described in other tissues of pigeons (MLSP, 35 years) compared to rats (MLSP, 4 years), indicate that oxidative damage to proteins, lipids and mitochondrial DNA are lower in birds (very

Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease: Why is it important and why is it hard?

DEFF Research Database (Denmark)

Nedić, Olgica; Rogowska-Wrzesinska, Adelina; Rattan, Suresh

2015-01-01

Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically...

Hypochlorite-induced oxidation of proteins in plasma

DEFF Research Database (Denmark)

Hawkins, C L; Davies, Michael Jonathan

1999-01-01

Activated phagocyte cells generate hypochlorite (HOCl) via the release of H2O2 and the enzyme myeloperoxidase. Plasma proteins are major targets for HOCl, although little information is available about the mechanism(s) of oxidation. In this study the reaction of HOCl (at least 50 microM) with dil......Activated phagocyte cells generate hypochlorite (HOCl) via the release of H2O2 and the enzyme myeloperoxidase. Plasma proteins are major targets for HOCl, although little information is available about the mechanism(s) of oxidation. In this study the reaction of HOCl (at least 50 micro......M) with diluted fresh human plasma has been shown to generate material that oxidizes 5-thio-2-nitrobenzoic acid; these oxidants are believed to be chloramines formed from the reaction of HOCl with protein amine groups. Chloramines have also been detected with isolated plasma proteins treated with HOCl. In both...... more efficient. The reaction of fresh diluted plasma with HOCl also gives rise to protein-derived nitrogen-centred radicals in a time- and HOCl-concentration-dependent manner; these have been detected by EPR spin trapping. Identical radicals have been detected with isolated HOCl-treated plasma proteins...

Plant cytoplasmic GAPDH: redox post-translational modifications and moonlighting properties

Directory of Open Access Journals (Sweden)

Mirko eZaffagnini

2013-11-01

Full Text Available Glyceraldehyde-3-phosphate dehydrogenase (GAPDH is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively-modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively-modified GAPDH in stress signaling pathways in plants.

Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

Energy Technology Data Exchange (ETDEWEB)

Inadomi, Chiaki, E-mail: inadomic@nagasaki-u.ac.jp [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Murata, Hiroaki [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Ihara, Yoshito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Biochemistry, Wakayama Medical University, Wakayama 641-8509 (Japan); Goto, Shinji; Urata, Yoshishige [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Yodoi, Junji [Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto 606-8507 (Japan); Kondo, Takahito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Sumikawa, Koji [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan)

2012-08-31

Highlights: Black-Right-Pointing-Pointer GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. Black-Right-Pointing-Pointer NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. Black-Right-Pointing-Pointer Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

Oxidation and thermal shock behavior of thermal barrier coated 18/10CrNi alloy with coating modifications

Energy Technology Data Exchange (ETDEWEB)

Guergen, Selim [Vocational School of Transportation, Anadolu University, Eskisehir (Turkmenistan); Diltemiz, Seyid Fehmi [Turkish Air Force1st Air Supply and Maintenance Center Command, Eskisehir (Turkmenistan); Kushan, Melih Cemal [Dept. of Mechanical Engineering, Eskisehir Osmangazi University, Eskisehir (Turkmenistan)

2017-01-15

In this study, substrates of 18/10CrNi alloy plates were initially sprayed with a Ni-21Cr-10Al-1Y bond coat and then with an yttria stabilized zirconia top coat by plasma spraying. Subsequently, plasma-sprayed Thermal barrier coatings (TBCs) were treated with two different modification methods, namely, vacuum heat treatment and laser glazing. The effects of modifications on the oxidation and thermal shock behavior of the coatings were evaluated. The effect of coat thickness on the bond strength of the coats was also investigated. Results showed enhancement of the oxidation resistance and thermal shock resistance of TBCs following modifications. Although vacuum heat treatment and laser glazing exhibited comparable results as per oxidation resistance, the former generated the best improvement in the thermal shock resistance of the TBCs. Bond strength also decreased as coat thickness increased.

Chemical synthesis of membrane proteins by the removable backbone modification method.

Science.gov (United States)

Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

2017-12-01

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Oxidant/Antioxidant Balance in Animal Nutrition and Health: The Role of Protein Oxidation

OpenAIRE

Celi, Pietro; Gabai, Gianfranco

2015-01-01

This review examines the role that oxidative stress (OS), and protein oxidation in particular, plays in nutrition, metabolism, and health of farm animals. The route by which redox homeostasis is involved in some important physiological functions and the implications of the impairment of oxidative status on animal health and diseases is also examined. Proteins have various and, at the same time, unique biological functions and their oxidation can result in structural changes and various functi...

Oxidant/antioxidant balance in animal nutrition and health: the role of protein oxidation

OpenAIRE

Pietro eCeli; Pietro eCeli; Gianfranco eGabai

2015-01-01

This review examines the role that oxidative stress, and protein oxidation in particular, plays in nutrition, metabolism and health of farm animals. The route by which redox homeostasis is involved in some important physiological functions and the implications of the impairment of oxidative status on animal health and diseases is also examined. Proteins have various and, at the same time, unique biological functions and their oxidation can result in structural changes and various functional m...

5,5'-Dithio-bis(2-nitrobenzoic acid) modification of cysteine improves the crystal quality of human chloride intracellular channel protein 2

International Nuclear Information System (INIS)

Mi Wei; Li Lanfen; Su Xiaodong

2008-01-01

Structural studies of human chloride intracellular channel protein 2 (CLIC2) had been hampered by the problem of generating suitable crystals primarily due to the protein containing exposed cysteines. Several chemical reagents were used to react with the cysteines on CLIC2 in order to modify the redox state of the protein. We have obtained high quality crystals that diffracted to better than 2.5 A at a home X-ray source by treating the protein with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). After solving the crystal structure of CLIC2, we found that the DTNB had reacted with the Cys 114 , and made CLIC2 in a homogenous oxidized state. This study demonstrated that the DTNB modification drastically improved the crystallization of CLIC2, and it implied that this method may be useful for other proteins containing exposed cysteines in general

Analysis of Protein-Phenolic Compound Modifications Using Electrochemistry Coupled to Mass Spectrometry.

Science.gov (United States)

Kallinich, Constanze; Schefer, Simone; Rohn, Sascha

2018-01-29

In the last decade, electrochemical oxidation coupled with mass spectrometry has been successfully used for the analysis of metabolic studies. The application focused in this study was to investigate the redox potential of different phenolic compounds such as the very prominent chlorogenic acid. Further, EC/ESI-MS was used as preparation technique for analyzing adduct formation between electrochemically oxidized phenolic compounds and food proteins, e.g., alpha-lactalbumin or peptides derived from a tryptic digestion. In the first step of this approach, two reactant solutions are combined and mixed: one contains the solution of the digested protein, and the other contains the phenolic compound of interest, which was, prior to the mixing process, electrochemically transformed to several oxidation products using a boron-doped diamond working electrode. As a result, a Michael-type addition led to covalent binding of the activated phenolic compounds to reactive protein/peptide side chains. In a follow-up approach, the reaction mix was further separated chromatographically and finally detected using ESI-HRMS. Compound-specific, electrochemical oxidation of phenolic acids was performed successfully, and various oxidation and reaction products with proteins/peptides were observed. Further optimization of the reaction (conditions) is required, as well as structural elucidation concerning the final adducts, which can be phenolic compound oligomers, but even more interestingly, quite complex mixtures of proteins and oxidation products.

Proteomic identification of early salicylate- and flg22-responsive redox-sensitive proteins in Arabidopsis

KAUST Repository

Liu, Peng

2015-02-27

Accumulation of reactive oxygen species (ROS) is one of the early defense responses against pathogen infection in plants. The mechanism about the initial and direct regulation of the defense signaling pathway by ROS remains elusive. Perturbation of cellular redox homeostasis by ROS is believed to alter functions of redox-sensitive proteins through their oxidative modifications. Here we report an OxiTRAQ-based proteomic study in identifying proteins whose cysteines underwent oxidative modifications in Arabidopsis cells during the early response to salicylate or flg22, two defense pathway elicitors that are known to disturb cellular redox homeostasis. Among the salicylate- and/or flg22-responsive redox-sensitive proteins are those involved in transcriptional regulation, chromatin remodeling, RNA processing, post-translational modifications, and nucleocytoplasmic shuttling. The identification of the salicylate-/flg22-responsive redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding biological significance of their oxidative modifications during the plant defense response.

Protein modification and replicative senescence of WI-38 human embryonic fibroblasts

DEFF Research Database (Denmark)

Ahmed, Emad K; Rogowska-Wrzesinska, Adelina; Roepstorff, Peter

2010-01-01

reflects a preferential accumulation of damaged proteins within the mitochondria during cellular senescence. Accumulation of AGE-modified proteins could be explained by the senescence-associated decreased activity of glyoxalase-I, the major enzyme involved in the detoxification of the glycating agents...... methylglyoxal and glyoxal, in both cytosol and mitochondria. This finding suggests a role of detoxification systems in the age-related build-up of damaged proteins. Moreover, the oxidized protein repair system methionine sulfoxide reductase was more affected in the mitochondria than in the cytosol during......Summary Oxidized proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro. To better understand the mechanisms by which these damaged proteins...

N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

Science.gov (United States)

Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

2015-01-01

The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

Measuring site occupancy: a new perspective on cysteine oxidation.

Science.gov (United States)

Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna; Williamson, James; Roepstorff, Peter

2014-10-01

Site occupancy is an extremely important aspect of quantification of protein modifications. Knowing the degree of modification of each oxidised cysteine residue is critical to understanding the biological role of these modifications. Yet modification site occupancy is very often overlooked, in part because there are very few analytical tools that allow such measurements. Here we present a new strategy, which provides quantitative analysis of cysteine S-nitrosylation (SNO) and S-sulfenylation (SOH) simultaneously at the resolution of single cysteine and allows for determination of relative oxidation occupancy of the modification site. We show that, on one hand, heavily modified cysteines are not necessarily involved in the response to oxidative stress. On the other hand residues with low modification level can be dramatically affected by mild oxidative imbalance. We make use of high resolution mass spectrometry. The method relies on differential reduction of "total" cysteines, SNO cysteines and SOH cysteines with TCEP, sodium ascorbate and sodium arsenite respectively followed by iodoTMT(TM) alkylation. Enrichment of iodoTMT(TM)-containing peptides is performed using anti-TMT antibody. In vivo model of mild oxidative stress in Escherichia coli is used. To induce endogenous SNO bacteria were grown anaerobically in minimal media supplemented with fumarate or nitrate. Short-term treatment with submilimolar levels of hydrogen peroxide were used to induce SOH. We have quantified 114 SNO/SOH modified peptides corresponding to 90 proteins. Only 6 modified peptides changed significantly under mild oxidative stress. Quantitative information allowed us to determine relative modification site occupancy of each identified modified residue and pin point heavily modified ones. The method proved to be precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale and brings a new perspective on the role of the modification site

The Role of S-Nitrosylation and S-Glutathionylation of Protein Disulphide Isomerase in Protein Misfolding and Neurodegeneration

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M. Halloran

2013-01-01

Full Text Available Neurodegenerative diseases involve the progressive loss of neurons, and a pathological hallmark is the presence of abnormal inclusions containing misfolded proteins. Although the precise molecular mechanisms triggering neurodegeneration remain unclear, endoplasmic reticulum (ER stress, elevated oxidative and nitrosative stress, and protein misfolding are important features in pathogenesis. Protein disulphide isomerase (PDI is the prototype of a family of molecular chaperones and foldases upregulated during ER stress that are increasingly implicated in neurodegenerative diseases. PDI catalyzes the rearrangement and formation of disulphide bonds, thus facilitating protein folding, and in neurodegeneration may act to ameliorate the burden of protein misfolding. However, an aberrant posttranslational modification of PDI, S-nitrosylation, inhibits its protective function in these conditions. S-nitrosylation is a redox-mediated modification that regulates protein function by covalent addition of nitric oxide- (NO- containing groups to cysteine residues. Here, we discuss the evidence for abnormal S-nitrosylation of PDI (SNO-PDI in neurodegeneration and how this may be linked to another aberrant modification of PDI, S-glutathionylation. Understanding the role of aberrant S-nitrosylation/S-glutathionylation of PDI in the pathogenesis of neurodegenerative diseases may provide insights into novel therapeutic interventions in the future.

Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds

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Michaela Mühlberg

2015-05-01

Full Text Available To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

Tyrosine Sulfation as a Protein Post-Translational Modification

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Yuh-Shyong Yang

2015-01-01

Full Text Available Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

Effects of UVB-induced oxidative stress on protein expression and specific protein oxidation in normal human epithelial keratinocytes: a proteomic approach

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De Marco Federico

2010-03-01

Full Text Available Abstract Background The UVB component of solar ultraviolet irradiation is one of the major risk factors for the development of skin cancer in humans. UVB exposure elicits an increased generation of reactive oxygen species (ROS, which are responsible for oxidative damage to proteins, DNA, RNA and lipids. In order to examine the biological impact of UVB irradiation on skin cells, we used a parallel proteomics approach to analyze the protein expression profile and to identify oxidatively modified proteins in normal human epithelial keratinocytes. Results The expression levels of fifteen proteins - involved in maintaining the cytoskeleton integrity, removal of damaged proteins and heat shock response - were differentially regulated in UVB-exposed cells, indicating that an appropriate response is developed in order to counteract/neutralize the toxic effects of UVB-raised ROS. On the other side, the redox proteomics approach revealed that seven proteins - involved in cellular adhesion, cell-cell interaction and protein folding - were selectively oxidized. Conclusions Despite a wide and well orchestrated cellular response, a relevant oxidation of specific proteins concomitantly occurs in UVB-irradiated human epithelial Keratinocytes. These modified (i.e. likely dysfunctional proteins might result in cell homeostasis impairment and therefore eventually promote cellular degeneration, senescence or carcinogenesis.

Prediction of protein post-translational modifications: main trends and methods

Science.gov (United States)

Sobolev, B. N.; Veselovsky, A. V.; Poroikov, V. V.

2014-02-01

The review summarizes main trends in the development of methods for the prediction of protein post-translational modifications (PTMs) by considering the three most common types of PTMs — phosphorylation, acetylation and glycosylation. Considerable attention is given to general characteristics of regulatory interactions associated with PTMs. Different approaches to the prediction of PTMs are analyzed. Most of the methods are based only on the analysis of the neighbouring environment of modification sites. The related software is characterized by relatively low accuracy of PTM predictions, which may be due both to the incompleteness of training data and the features of PTM regulation. Advantages and limitations of the phylogenetic approach are considered. The prediction of PTMs using data on regulatory interactions, including the modular organization of interacting proteins, is a promising field, provided that a more carefully selected training data will be used. The bibliography includes 145 references.

A Nucleocytoplasmic Shuttling Protein in Oxidative Stress Tolerance

Energy Technology Data Exchange (ETDEWEB)

Ow, David W.; Song, Wen

2003-03-26

Plants for effective extraction of toxic metals and radionuclides must tolerate oxidative stress. To identify genes that enhance oxidative stress tolerance, an S. pombe cDNA expression plasmid library was screened for the ability to yield hypertolerant colonies. Here, we report on the properties of one gene that confers hypertolerance to cadmium and oxidizing chemicals. This gene appears to be conserved in other organisms as homologous genes are found in human, mouse, fruitfly and Arabidopsis. The fruitfly and Arabidopsis genes likewise enhance oxidative stress tolerance in fission yeast. During oxidative stress, the amount of mRNA does not change, but protein fusions to GFP relocate from the cytoplasm to the nucleus. The same pattern is observed with the Arabidopsis homologue-GFP fusion protein. This behavior suggests a signaling role in oxidative stress tolerance and these conserved proteins may be targets for engineering stress tolerant plants for phytoremediation.

Potential disruption of protein-protein interactions by graphene oxide

International Nuclear Information System (INIS)

Feng, Mei; Kang, Hongsuk; Luan, Binquan; Yang, Zaixing; Zhou, Ruhong

2016-01-01

Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

Potential disruption of protein-protein interactions by graphene oxide

Energy Technology Data Exchange (ETDEWEB)

Feng, Mei [Department of Physics, Institute of Quantitative Biology, Zhejiang University, Hangzhou 310027 (China); Kang, Hongsuk; Luan, Binquan [Computational Biological Center, IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598 (United States); Yang, Zaixing [Institute of Quantitative Biology and Medicine, SRMP and RAD-X, and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123 (China); Zhou, Ruhong, E-mail: ruhong@us.ibm.com [Department of Physics, Institute of Quantitative Biology, Zhejiang University, Hangzhou 310027 (China); Computational Biological Center, IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598 (United States); Department of Chemistry, Columbia University, New York, New York 10027 (United States)

2016-06-14

Graphene oxide (GO) is a promising novel nanomaterial with a wide range of potential biomedical applications due to its many intriguing properties. However, very little research has been conducted to study its possible adverse effects on protein-protein interactions (and thus subsequent toxicity to human). Here, the potential cytotoxicity of GO is investigated at molecular level using large-scale, all-atom molecular dynamics simulations to explore the interaction mechanism between a protein dimer and a GO nanosheet oxidized at different levels. Our theoretical results reveal that GO nanosheet could intercalate between the two monomers of HIV-1 integrase dimer, disrupting the protein-protein interactions and eventually lead to dimer disassociation as graphene does [B. Luan et al., ACS Nano 9(1), 663 (2015)], albeit its insertion process is slower when compared with graphene due to the additional steric and attractive interactions. This study helps to better understand the toxicity of GO to cell functions which could shed light on how to improve its biocompatibility and biosafety for its wide potential biomedical applications.

Protein oxidation and degradation caused by particulate matter

Science.gov (United States)

Lai, Ching-Huang; Lee, Chun-Nin; Bai, Kuan-Jen; Yang, You-Lan; Chuang, Kai-Jen; Wu, Sheng-Ming; Chuang, Hsiao-Chi

2016-09-01

Particulate matter (PM) modulates the expression of autophagy; however, the role of selective autophagy by PM remains unclear. The objective of this study was to determine the underlying mechanisms in protein oxidation and degradation caused by PM. Human epithelial A549 cells were exposed to diesel exhaust particles (DEPs), urban dust (UD), and carbon black (CB; control particles). Cell survival and proliferation were significantly reduced by DEPs and UD in A549 cells. First, benzo(a)pyrene diolepoxide (BPDE) protein adduct was caused by DEPs at 150 μg/ml. Methionine oxidation (MetO) of human albumin proteins was induced by DEPs, UD, and CB; however, the protein repair mechanism that converts MetO back to methionine by methionine sulfoxide reductases A (MSRA) and B3 (MSRB3) was activated by DEPs and inhibited by UD, suggesting that oxidized protein was accumulating in cells. As to the degradation of oxidized proteins, proteasome and autophagy activation was induced by CB with ubiquitin accumulation, whereas proteasome and autophagy activation was induced by DEPs without ubiquitin accumulation. The results suggest that CB-induced protein degradation may be via an ubiquitin-dependent autophagy pathway, whereas DEP-induced protein degradation may be via an ubiquitin-independent autophagy pathway. A distinct proteotoxic effect may depend on the physicochemistry of PM.

Surface modification of graphene oxide nanosheets by protamine sulfate/sodium alginate for anti-cancer drug delivery application

Science.gov (United States)

Xie, Meng; Zhang, Feng; Liu, Lijiao; Zhang, Yanan; Li, Yeping; Li, Huaming; Xie, Jimin

2018-05-01

In order to improve the efficiency of anticancer drug delivery, a graphene oxide (GO) based drug delivery system modificated by natural peptide protamine sulfate (PRM) and sodium alginate (SA) was established via electrostatic attraction at each step of adsorption based on layer-by-layer self-assembly. The nanocomposites were then loaded with anticancer drug doxorubicin hydrochloride (DOX) to estimate the feasibility as drug carriers. The nanocomposites loaded with DOX revealed a remarkable pH-sensitive drug release property. The modification with protamine sulfate and sodium alginate could not only impart the nanocomposites an improved dispersibility and stability under physiological pH, but also suppress the protein adhesion. Due to the high water dispersibility and the small particle size, GO-PRM/SA nanocomposites were able to be uptaken by MCF-7 cells. It was found that GO-PRM/SA nanocomposites exhibited no obvious cytotoxicity towards MCF-7 cells, while GO-PRM/SA-DOX exhibited better cytotoxicity than GO-DOX. Therefore, the GO-PRM/SA nanocomposites were feasible as drug delivery vehicles.

Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide

International Nuclear Information System (INIS)

Ohno, Kazuki; Okuda, Kosaku; Uehara, Takashi

2015-01-01

Highlights: • PTEN is S-sulfhydrated endogenously in SH-SY5Y human neuroblastoma cells. • Preventing this modification by knocking down CBS renders PTEN sensitive to NO. • pAkt levels are increased significantly in CBS siRNA-transfected cells. • H 2 S functions as an endogenous regulator of PTEN in neuronal cells. - Abstract: Hydrogen sulfide (H 2 S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H 2 S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H 2 S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H 2 S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H 2 S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions

Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide

Energy Technology Data Exchange (ETDEWEB)

Ohno, Kazuki; Okuda, Kosaku; Uehara, Takashi, E-mail: uehara@pharm.okayama-u.ac.jp

2015-01-02

Highlights: • PTEN is S-sulfhydrated endogenously in SH-SY5Y human neuroblastoma cells. • Preventing this modification by knocking down CBS renders PTEN sensitive to NO. • pAkt levels are increased significantly in CBS siRNA-transfected cells. • H{sub 2}S functions as an endogenous regulator of PTEN in neuronal cells. - Abstract: Hydrogen sulfide (H{sub 2}S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H{sub 2}S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H{sub 2}S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H{sub 2}S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H{sub 2}S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.

Maillard-reaction-induced modification and aggregation of proteins and hardening of texture in protein bar model systems.

Science.gov (United States)

Zhou, Peng; Guo, Mufan; Liu, Dasong; Liu, Xiaoming; Labuza, Teodore P

2013-03-01

The hardening of high-protein bars causes problems in their acceptability to consumers. The objective of this study was to determine the progress of the Maillard reaction in model systems of high-protein nutritional bars containing reducing sugars, and to illustrate the influences of the Maillard reaction on the modification and aggregation of proteins and the hardening of bar matrices during storage. The progress of the Maillard reaction, glycation, and aggregation of proteins, and textural changes in bar matrices were investigated during storage at 25, 35, and 45 °C. The initial development of the Maillard reaction caused little changes in hardness; however, further storage resulted in dramatic modification of protein with formation of high-molecular-weight polymers, resulting in the hardening in texture. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula minimized the changes in texture. The hardening of high-protein bars causes problems in their acceptability to consumers. Maillard reaction is one of the mechanisms contributing to the hardening of bar matrix, particularly for the late stage of storage. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula will minimize the changes in texture. © 2013 Institute of Food Technologists®

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

Science.gov (United States)

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

Mining Proteomic Data to Expose Protein Modifications in Methanosarcina mazei strain Gö1

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Deborah eLeon

2015-03-01

Full Text Available Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material. Strategies to mine information from these discards are presented, along with discussion of features that, when present, provide strong support for modifications. In this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154 proteins. Many of the observed proteins displayed post-translationally modified forms, including O-formylated and methyl-esterified segments that appear biologically relevant (i.e., not artifacts of sample handling. Interesting cleavages and modifications (e.g., S-cyanylation and trimethylation were observed near catalytic sites of methanogenesis enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A orthologue, MA0829, underwent signal peptide excision. Experimental results contrast with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976 were found to be glycosylated, and employing chromatography tailored specifically for glycopeptides will likely reveal more.This study supplements limited, existing experimental datasets of mature archaeal N-termini, including presence or absence of signal peptides, translation initiation sites, and other processing. Methanosarcina surface and membrane proteins are richly modified.

Surface modification and functionalization of metal and metal oxide nanoparticles by organic ligands

NARCIS (Netherlands)

Neouze, M.A.; Schubert, U.S.

2008-01-01

Metal or metal oxide nanoparticles possess unique features compared to equivalent larger-scale materials. For applications, it is often necessary to stabilize or functionalize such nanoparticles. Thus, modification of the surface of nanoparticles is an important chemical challenge. In this survey,

MODIFICATION OF ERYTHROCYTE MEMBRANE PROTEINS WITH POLYETHYLENE GLYCOL 1500

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N. G. Zemlianskykh

2016-10-01

Full Text Available The aim of the work was to study the effect of polyethylene glycol PEG-1500 on the Ca2+-ATPase activity and changes in CD44 surface marker expression in human erythrocyte membranes. Determination of the Ca2+-ATPase activity was carried out in sealed erythrocyte ghosts by the level of accumulation of inorganic phosphorus. Changes in the expression of CD44 and amount of CD44+-erythrocytes were evaluated by flow cytometry. The inhibition of Ca2+-ATPase activity and a reduction in the level of CD44 expression and also the decrease in the amount CD44+-cells were found, reflecting a fairly complex restructuring in the membrane-cytoskeleton complex of erythrocytes under the influence of PEG-1500. Effect of PEG-1500 on the surface CD44 marker could be mediated by modification of proteins of membrane-cytoskeleton complex, as indicated by accelerated loss of CD44 in erythrocyte membranes after application of protein cross-linking reagent diamide. Reduced activity of Ca2+-ATPase activity may contribute to the increase in intracellular Ca2+ level and thus leads to a modification of interactions of integral proteins with cytoskeletal components that eventually could result in membrane vesiculation and decreasing in expression of the CD44 marker, which is dynamically linked to the cytoskeleton.

Reversible oxidative modification: a key mechanism of Na+-K+ pump regulation.

Science.gov (United States)

Figtree, Gemma A; Liu, Chia-Chi; Bibert, Stephanie; Hamilton, Elisha J; Garcia, Alvaro; White, Caroline N; Chia, Karin K M; Cornelius, Flemming; Geering, Kaethi; Rasmussen, Helge H

2009-07-17

Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na(+)-K(+) pump via protein kinase (PK)C-dependent activation of NADPH oxidase. We examined whether this is mediated by oxidative modification of the pump subunits. We detected glutathionylation of beta(1), but not alpha(1), subunits in rabbit ventricular myocytes at baseline. beta(1) Subunit glutathionylation was increased by peroxynitrite (ONOO(-)), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased alpha(1)/beta(1) subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with beta(1) subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na(+)-K(+) pump current (I(p)). Cysteine (Cys46) of the beta(1) subunit was the likely candidate for glutathionylation. We expressed Na(+)-K(+) pump alpha(1) subunits with wild-type or Cys46-mutated beta(1) subunits in Xenopus oocytes. ONOO(-) induced glutathionylation of beta(1) subunit and a decrease in Na(+)-K(+) pump turnover number. This was eliminated by mutation of Cys46. ONOO(-) also induced glutathionylation of the Na(+)-K(+) ATPase beta(1) subunit from pig kidney. This was associated with a approximately 2-fold decrease in the rate-limiting E(2)-->E(1) conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na(+)-K(+) pump occurs via glutathionylation of its beta(1) subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na(+) levels.

The Human Thioredoxin System: Modifications and Clinical Applications

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Seyed Isaac Hashemy

2011-03-01

Full Text Available The thioredoxin system, comprising thioredoxin (Trx, thioredoxin reductase (TrxR and NADPH, is one of the major cellular antioxidant systems, implicated in a large and growing number of biological functions. Trx acts as an oxidoreductase via a highly conserved dithiol/disulfide motif located in the active site (-Trp-Cys-Gly-Pro-Cys-Lys-. Different factors are involved in the regulation of Trx activity, including its expression level, localization, protein-protein interactions, post-translational modifications and some chemical inhibitors. Mammalian TrxRs are selenoproteins which have a –Cys-Val-Asn-Val-Gly-Cys- N-terminal active site, as well as a C-terminal selenium-containing active site. Besides two Cys-residues in the redox-regulatory domain of cytosolic Trx (Trx1, human Trx1 has three additional Cys-residues. Post-translational modifications of human Trx1 which are involved in the regulation of its activity can happen via modification of Cys-residues including thiol oxidation, glutathionylation and S-nitrosylation or via modification of other amino acid residues such as nitration of Tyr-49. Because of the numerous functions of the thioredoxin system, its inhibition (mainly happens via the targeting TrxR can result in major cellular consequences, which are potentially pro-oxidant in nature, leading to cell death via necrosis or apoptosis if overexpression of Trx and other antioxidative enzymes can not recuperate cell response. Considering this feature, several anticancer drugs have been used which can inhibit TrxR. Elevated levels of Trx and/or TrxR have been reported in many different human malignancies, positively correlated with aggressive tumor growth and poor prognosis. Moreover, anti-oxidative and anti-apoptotic effects of Trx are reasons to study its clinical application as a drug.

Copper-mediated oxidative degradation of catecholamines and oxidative damage of protein

Energy Technology Data Exchange (ETDEWEB)

Goncalves, P.R.; Harria, M.I.N.; Felix, J.M.; Hoffmann, M.E. [Universidade Estadual de Campinas, SP (Brazil). Inst. de Biologia

1997-12-31

Full text. Degradative oxidation of catecholamines has been a matter of large interest in recent years due to the evidences associating their autoxidation with the etiology of neurotoxic and cardiotoxic processes. In this work we present data on the degradative oxidation of catecholamines of physiological importance: isoproterenol (IP), epinephrine (EP), norepinephrine (NEP), deoxyepinephrine (DEP) and dopamine (DA). The degradative oxidation of the catecholamines was followed by measurement of spectral changes and oxygen consumption by neutral aqueous solutions. The data show that Cu{sup 2+} strongly accelerated the rate of catecholamine oxidation, following the decreasing order; EP>DEP>IP>NEP>DA. The production of superoxide anion radical during catecholamine oxidation was very slow, even in the presence of Cu{sup 2+}. The ability of IP to induce damages on bovine serum albumin (BSA) was determined by measuring the formation of carbonyl-groups in the protein, detected by reduction with tritiated Na BH{sub 4}. The incubation of BSA with IP (50-500{mu}M), in the presence of 100{mu}M Cu{sup 2+} leaded to an increased and dose dependent {sup 3} H-incorporation by the oxidized protein. The production of oxidative damage by IP/Cu{sup 2+} was accompanied by marked BSA fragmentation, detected by SDS-polyacrylamide gel dependent (25-400{mu}M IP) des appearance of the original BSA band and appearance of smaller fragments spread in the gel, when incubation has been done in the presence of 100{mu}M Cu{sup 2+}. These results suggest that copper-catalysed oxidative degradation of proteins induced by catecholamines might be critically involved in the toxic action of these molecules

Genetic Variation and Its Reflection on Posttranslational Modifications in Frequency Clock and Mating Type a-1 Proteins in Sordaria fimicola

OpenAIRE

Arif, Rabia; Akram, Faiza; Jamil, Tazeen; Mukhtar, Hamid; Lee, Siu Fai; Saleem, Muhammad

2017-01-01

Posttranslational modifications (PTMs) occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment o...

In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells.

Science.gov (United States)

Espino, Jessica A; Mali, Vishaal S; Jones, Lisa M

2015-08-04

Protein footprinting coupled with mass spectrometry has become a widely used tool for the study of protein-protein and protein-ligand interactions and protein conformational change. These methods provide residue-level analysis on protein interaction sites and have been successful in studying proteins in vitro. The extension of these methods for in cell footprinting would open an avenue to study proteins that are not amenable for in vitro studies and would probe proteins in their native environment. Here we describe the application of an oxidative-based footprinting approach inside cells in which hydroxyl radicals are used to oxidatively modify proteins. Mass spectrometry is used to detect modification sites and to calculate modification levels. The method is probing biologically relevant proteins in live cells, and proteins in various cellular compartments can be oxdiatively modified. Several different amino acid residues are modified making the method a general labeling strategy for the study of a variety of proteins. Further, comparison of the extent of oxidative modification with solvent accessible surface area reveals the method successfully probes solvent accessibility. This marks the first time protein footprinting has been performed in live cells.

Prediction of protein modification sites of pyrrolidone carboxylic acid using mRMR feature selection and analysis.

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Lu-Lu Zheng

Full Text Available Pyrrolidone carboxylic acid (PCA is formed during a common post-translational modification (PTM of extracellular and multi-pass membrane proteins. In this study, we developed a new predictor to predict the modification sites of PCA based on maximum relevance minimum redundancy (mRMR and incremental feature selection (IFS. We incorporated 727 features that belonged to 7 kinds of protein properties to predict the modification sites, including sequence conservation, residual disorder, amino acid factor, secondary structure and solvent accessibility, gain/loss of amino acid during evolution, propensity of amino acid to be conserved at protein-protein interface and protein surface, and deviation of side chain carbon atom number. Among these 727 features, 244 features were selected by mRMR and IFS as the optimized features for the prediction, with which the prediction model achieved a maximum of MCC of 0.7812. Feature analysis showed that all feature types contributed to the modification process. Further site-specific feature analysis showed that the features derived from PCA's surrounding sites contributed more to the determination of PCA sites than other sites. The detailed feature analysis in this paper might provide important clues for understanding the mechanism of the PCA formation and guide relevant experimental validations.

Myofibrillar protein oxidation affects filament charges, aggregation and water-holding

NARCIS (Netherlands)

Bao, Yulong; Boeren, Sjef; Ertbjerg, Per

2018-01-01

Hypochlorous acid (HClO) is a strong oxidant that is able to mediate protein oxidation. In order to study the effect of oxidation on charges, aggregation and water-holding of myofibrillar proteins, extracted myofibrils were oxidized by incubation with different concentrations of HClO (0, 1, 5,

[Characteristics of proteins synthesized by hydrogen-oxidizing microorganisms].

Science.gov (United States)

Volova, T G; Barashkov, V A

2010-01-01

The study was conducted to determine the biological value of proteins synthesized by hydrogen-oxidizing microorganisms--the hydrogen bacteria Alcaligenes eutrophus Z1 and Ralstonia eutropha B5786 and the CO-resistant strain of carboxydobacterium Seliberia carboxydohydrogena Z1062. Based on a number of significant parameters characterizing the biological value of a product, the proteins of hydrogen-oxidizing microorganisms have been found to occupy an intermediate position between traditional animal and plant proteins. The high total protein in biomass of these microorganisms, their complete amino acid content, and availability to proteolytic enzymes allow for us to consider these microorganisms as potential protein producers.

Prediction of human protein function from post-translational modifications and localization features

DEFF Research Database (Denmark)

Jensen, Lars Juhl; Gupta, Ramneek; Blom, Nikolaj

2002-01-01

a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects......We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from...

Modification of Coal Char-loaded TiO2 by Sulfonation and Alkylsilylation to Enhance Catalytic Activity in Styrene Oxidation with Hydrogen Peroxide as Oxidant

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Mukhamad Nurhadi

2017-04-01

Full Text Available The modified coal char from low-rank coal by sulfonation, titanium impregnation and followed by alkyl silylation possesses high catalytic activity in styrene oxidation. The surface of coal char was undergone several steps as such: modification using concentrated sulfuric acid in the sulfonation process, impregnation of 500 mmol titanium(IV isopropoxide and followed by alkyl silylation of n-octadecyltriclorosilane (OTS. The catalysts were characterized by X-ray diffraction (XRD, IR spectroscopy, nitrogen adsorption, and hydrophobicity. The catalytic activity of the catalysts has been examined in the liquid phase styrene oxidation by using aqueous hydrogen peroxide as oxidant. The catalytic study showed the alkyl silylation could enhance the catalytic activity of Ti-SO3H/CC-600(2.0. High catalytic activity and reusability of the o-Ti-SO3H/CC-600(2.0 were related to the modification of local environment of titanium active sites and the enhancement the hydrophobicity of catalyst particle by alkyl silylation. Copyright © 2017 BCREC GROUP. All rights reserved Received: 24th May 2016; Revised: 11st October 2016; Accepted: 18th October 2016 How to Cite: Nurhadi, M. (2017. Modification of Coal Char-loaded TiO2 by Sulfonation and Alkylsilylation to Enhance Catalytic Activity in Styrene Oxidation with Hydrogen Peroxide as Oxidant. Bulletin of Chemical Reaction Engineering & Catalysis, 12 (1: 55-61 (doi:10.9767/bcrec.12.1.501.55-61 Permalink/DOI: http://dx.doi.org/10.9767/bcrec.12.1.501.55-61

Modification of radiation-induced oxidative damage in liposomal and microsomal membrane by eugenol

Energy Technology Data Exchange (ETDEWEB)

Pandey, B.N. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Lathika, K.M. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Mishra, K.P. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India)]. E-mail: kpm@magnum.barc.ernet.in

2006-03-15

Radiation-induced membrane oxidative damage, and their modification by eugenol, a natural antioxidant, was investigated in liposomes and microsomes. Liposomes prepared with DPH showed decrease in fluorescence after {gamma}-irradiation, which was prevented significantly by eugenol and correlated with magnitude of oxidation of phospholipids. Presence of eugenol resulted in substantial inhibition in MDA formation in irradiated liposomes/microsomes, which was less effective when added after irradiation. Similarly, the increase in phospholipase C activity observed after irradiation in microsomes was inhibited in samples pre-treated with eugenol. Results suggest association of radio- oxidative membrane damage with alterations in signaling molecules, and eugenol significantly prevented these membrane damaging events.

Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry

Science.gov (United States)

Souda, Puneet; Ryan, Christopher M.; Cramer, William A.; Whitelegge, Julian

2011-01-01

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein’s native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electroncapture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. PMID:21982782

Lysine-Directed Post-translational Modifications of Tau Protein in Alzheimer's Disease and Related Tauopathies

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Christiana Kontaxi

2017-08-01

Full Text Available Tau is a microtubule-associated protein responsible mainly for stabilizing the neuronal microtubule network in the brain. Under normal conditions, tau is highly soluble and adopts an “unfolded” conformation. However, it undergoes conformational changes resulting in a less soluble form with weakened microtubule stabilizing properties. Altered tau forms characteristic pathogenic inclusions in Alzheimer's disease and related tauopathies. Although, tau hyperphosphorylation is widely considered to be the major trigger of tau malfunction, tau undergoes several post-translational modifications at lysine residues including acetylation, methylation, ubiquitylation, SUMOylation, and glycation. We are only beginning to define the site-specific impact of each type of lysine modification on tau biology as well as the possible interplay between them, but, like phosphorylation, these modifications are likely to play critical roles in tau's normal and pathobiology. This review summarizes the latest findings focusing on lysine post-translational modifications that occur at both endogenous tau protein and pathological tau forms in AD and other tauopathies. In addition, it highlights the significance of a site-dependent approach of studying tau post-translational modifications under normal and pathological conditions.

Subcritical Water Hydrolysis of Peptides: Amino Acid Side-Chain Modifications

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Powell, Thomas; Bowra, Steve; Cooper, Helen J.

2017-09-01

Previously we have shown that subcritical water may be used as an alternative to enzymatic digestion in the proteolysis of proteins for bottom-up proteomics. Subcritical water hydrolysis of proteins was shown to result in protein sequence coverages greater than or equal to that obtained following digestion with trypsin; however, the percentage of peptide spectral matches for the samples treated with trypsin were consistently greater than for those treated with subcritical water. This observation suggests that in addition to cleavage of the peptide bond, subcritical water treatment results in other hydrolysis products, possibly due to modifications of amino acid side chains. Here, a model peptide comprising all common amino acid residues (VQSIKCADFLHYMENPTWGR) and two further model peptides (VCFQYMDRGDR and VQSIKADFLHYENPTWGR) were treated with subcritical water with the aim of probing any induced amino acid side-chain modifications. The hydrolysis products were analyzed by direct infusion electrospray tandem mass spectrometry, either collision-induced dissociation or electron transfer dissociation, and liquid chromatography collision-induced dissociation tandem mass spectrometry. The results show preferential oxidation of cysteine to sulfinic and sulfonic acid, and oxidation of methionine. In the absence of cysteine and methionine, oxidation of tryptophan was observed. In addition, water loss from aspartic acid and C-terminal amidation were observed in harsher subcritical water conditions. [Figure not available: see fulltext.

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

Science.gov (United States)

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.

Protein cysteine oxidation in redox signaling

DEFF Research Database (Denmark)

Forman, Henry Jay; Davies, Michael J; Krämer, Anna C

2017-01-01

Oxidation of critical signaling protein cysteines regulated by H2O2 has been considered to involve sulfenic acid (RSOH) formation. RSOH may subsequently form either a sulfenyl amide (RSNHR') with a neighboring amide, or a mixed disulfide (RSSR') with another protein cysteine or glutathione. Previ...

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

Science.gov (United States)

Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

2016-02-15

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

Science.gov (United States)

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.

Curcumin prevents the oxidation and lipid modification of LDL and its inhibition of prostacyclin generation by endothelial cells in culture.

Science.gov (United States)

Mahfouz, Mohamedain M; Zhou, Sherry Q; Kummerow, Fred A

2009-11-01

Low-density lipoprotein (LDL) was isolated from human plasma and oxidized by 5microM copper sulfate for 4h at 37 degrees C in the absence and presence of 1, 3, 5, 10, or 20microM of curcumin. LDL oxidized in the absence of curcumin (oxLDL) showed an increased levels of conjugated dienes, lipid peroxides (TBARS) and lysolecithin (lysoPC) and a significant loss of polyunsaturated fatty acids (PUFA). LDL oxidized with 5microM copper sulfate in the presence of curcumin caused a significant decrease of conjugated diene, lipid peroxides, lysoPC and significant increase of PUFA compared to oxLDL. These changes were dose dependent and reached a maximum at 5microM curcumin. Incubation of human endothelial cells (EC) with 200microg protein/ml of oxLDL caused a significant decrease of prostacyclin (PGI(2)) generation. LDL oxidized in presence of 5microM curcumin did not show any inhibition of PGI(2) generation compared to the control cells. These results indicate that curcumin is an effective chain-breaking antioxidant which prevents oxidation and lipid modification of LDL. The inhibition of oxLDL on PGI(2) is considered a contributing factor in the pathogenesis of thrombosis and atherosclerosis. Curcumin supplementation could be an effective strategy in preventing LDL oxidation and its impact on atherosclerosis and lesion formation.

Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids

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Ana Crnković

2018-02-01

Full Text Available Synthesis of proteins with noncanonical amino acids (ncAAs enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs and tRNAs (o-tRNAs. Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep. The incorporation of Sep into a green fluorescent protein (GFP in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs.

Modification of DNA radiolysis by DNA-binding proteins: Structural aspects

International Nuclear Information System (INIS)

Davidkova, M.; Stisova, V.; Goffinont, S.; Gillard, N.; Castaing, B.; Spotheim-Maurizot, M.

2006-01-01

Formation of specific complexes between proteins and their cognate DNA modulates the yields and the location of radiation damage on both partners of the complex. The radiolysis of DNA-protein complexes is studied for: (1) the Escherichia coli lactose operator-repressor complex, (2) the complex between DNA bearing an analogue of an abasic site and the repair protein Fpg of Lactococcus lactis. Experimental patterns of DNA damages are presented and compared to predicted damage distribution obtained using an improved version of the stochastic model RADACK. The same method is used for predicting the location of damages on the proteins. At doses lower than a threshold that depends on the system, proteins protect their specific binding site on DNA while at high doses, the studied complexes are disrupted mainly through protein damage. The loss of binding ability is the functional consequence of the amino-acids modification by OH . radicals. Many of the most probably damaged amino acids are essential for the DNA-protein interaction and within a complex are protected by DNA. (authors)

Differential plasma protein binding to metal oxide nanoparticles

International Nuclear Information System (INIS)

Deng, Zhou J; Mortimer, Gysell; Minchin, Rodney F; Schiller, Tara; Musumeci, Anthony; Martin, Darren

2009-01-01

Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO 2 , the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO 2 and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.

Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

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Li, R; Kast, J

2017-01-01

Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics. © 2017 Elsevier Inc. All rights reserved.

Green tea extract impairs meat emulsion properties by disturbing protein disulfide cross-linking.

Science.gov (United States)

Jongberg, Sisse; Terkelsen, Linda de S; Miklos, Rikke; Lund, Marianne N

2015-02-01

The dose-dependent effects of green tea extract (100, 500, or 1500ppm) on the textural and oxidative stability of meat emulsions were investigated, and compared to a control meat emulsion without extract. All levels of green tea extract inhibited formation of TBARS as a measure for lipid oxidation. Overall protein thiol oxidation and myosin heavy chain (MHC) cross-linking were inhibited by 100ppm green tea extract without jeopardizing the textural stability, while increasing concentrations of extract resulted in reduced thiol concentration and elevated levels of non-reducible protein modifications. Addition of 1500ppm green tea extract was found to modify MHC as evaluated by SDS-PAGE combining both protein staining and specific thiol staining, indicating that protein modifications generated through reactions of green tea phenolic compounds with protein thiols, disrupted the meat emulsion properties leading to reduced water holding capacity and textural stability. Hence, a low dose of green tea extract preserves both the textural and the oxidative stability of the meat proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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 Halotyrosine Antibody that Detects Increased Protein Modifications in Asthma Patients

Energy Technology Data Exchange (ETDEWEB)

Jin, Hongjun; Hallstrand, Teal S.; Daly, Don S.; Matzke, Melissa M.; Nair, Parameswaran; Bigelow, Diana J.; Pounds, Joel G.; Zangar, Richard C.

2014-01-31

Background-Airway inflammation plays an important pathophysiological role in asthma. Eosinophils produce hypobromite and bromotyrosine while neutrophils produce hypochlorite and chlorotyrosine. Objective-To evaluate halotyrosine modifications of individual airway proteins as a marker of inflammation in asthma using an antibody-based assay. Methods-We developed a novel monoclonal antibody (BTK-94C) that binds halogenated tyrosine residues, and used this antibody in a custom enzyme-linked immunosorbent assay (ELISA) microarray platform to examine halotyrosine levels in 23 proteins in three independent sets of sputum samples (52 samples total). Results-In 15 subjects with either no asthma, or with asthma characterized by high or low sputum eosinophil counts, we found associations between increased halotyrosine levels of at least three proteins and severity of airway hyperresponsiveness (AHR). Treatment with mepolizumab in 17 patients with sputum eosinophilia markedly reduced the sputum eosinophilia and significantly reduced halotyrosine levels in one sputum protein. Further analysis of 10 subjects with neutrophilic asthma and 10 health controls demonstrated a broad increase in halotyrosine in the patients with airway neutrophilia. Conclusions-Significantly higher levels of halotyrosine are associated with asthma in the asthma phenotypes we examined. The halotyrosine levels correlated with indirect AHR in the form of exercise-induced bronchoconstriction. Clinical Implication-An antibody-based assay for tyrosine halogenation in specific proteins may prove useful for assessing airway inflammation in asthma. Capsule Summary-An antibody to measure protein monobrominated tyrosine and other halotyrosine modifications was developed and used to evaluate halogenation in specific proteins in the airways for the first time. Associations were found between levels of halotyrosine and exercise-induced bronchoconstriction, and eosinophil and neutrophil inflammation in sputum from

Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects.

Science.gov (United States)

Batthyány, Carlos; Bartesaghi, Silvina; Mastrogiovanni, Mauricio; Lima, Analía; Demicheli, Verónica; Radi, Rafael

2017-03-01

"Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function. Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation. The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.

Modification of graphite structure by irradiation, revealed by thermal oxidation. Examination by electronic microscopy

International Nuclear Information System (INIS)

Rouaud, Michel

1969-01-01

Based on the analysis of images obtained by electronic microscopy, this document reports the comparative study of the action of neutrons on three different graphites: a natural one (Ticonderoga) and two pyrolytic ones (Carbone-Lorraine and Raytheon). The approach is based on the modification of features of thermal oxidation of graphites by dry air after irradiation. Different corrosion features are identified. The author states that there seems to be a relationship between the number and shape of these features, and defects existing on the irradiated graphite before oxidation. For low doses, the feature aspect varies with depth at which oxidation occurs. For higher doses, the aspect remains the same [fr

Oxidation of DNA, proteins and lipids by DOPA, protein-bound DOPA, and related catechol(amine)s

DEFF Research Database (Denmark)

Pattison, David I; Dean, Roger T; Davies, Michael Jonathan

2002-01-01

Incubation of free 3,4-dihydroxyphenylalanine (DOPA), protein-bound DOPA (PB-DOPA) and related catechols with DNA, proteins and lipids has been shown to result in oxidative damage to the target molecule. This article reviews these reactions with particular emphasis on those that occur in the pres......Incubation of free 3,4-dihydroxyphenylalanine (DOPA), protein-bound DOPA (PB-DOPA) and related catechols with DNA, proteins and lipids has been shown to result in oxidative damage to the target molecule. This article reviews these reactions with particular emphasis on those that occur...... in the presence of molecular O(2) and redox-active metal ions (e.g. Fe(3+), Cu(2+), Cr(6+)), which are known to increase the rate of DOPA oxidation. The majority of oxidative damage appears to be mediated by reactive oxygen species (ROS) such as superoxide and HO(.) radicals, though other DOPA oxidation products...

Comparison of protein degradation, protein oxidation, and μ-calpain activation between pale, soft, and exudative and red, firm, and nonexudative pork during postmortem aging.

Science.gov (United States)

Yin, Y; Zhang, W G; Zhou, G H; Guo, B

2014-08-01

The objective of this study was to investigate the differences in protein modifications between pale, soft, and exudative (PSE) and red, firm, and nonexudative (RFN) pork during postmortem (PM) aging. Longissimus dorsi (LD) including 8 PSE and 8 RFN muscles were individually removed from 16 carcasses. These 16 LD muscles were vacuum packaged at 24 h after slaughter and stored at 4°C for 1, 3, and 5 d. The centrifugation loss, drip loss, color, protein solubility, protein oxidation, protein degradation including desmin, troponin T, and integrin, and μ-calpain activation were determined. The pH of PSE samples was significantly lower than that of RFN samples at both 1 and 24 h PM (P 0.05). In addition, PSE pork presented a lower solubility of sarcoplasmic protein, myofibrillar protein, and total protein than RFN pork except the solubility of myofibrillar protein at d 1 (P firm, and nonexudative pork presented lower intensity of intact 80 kDa calpain and greater intensity of autolyzed 76 kDa product compared to PSE pork (P < 0.01). The results indicate that the degree of μ-calpain activation, the extent of protein degradation including desmin and integrin, and the level of protein solubility in PSE pork could contribute to its low water holding capacity during PM storage.

Photo-oxidation of proteins and its role in cataractogenesis

DEFF Research Database (Denmark)

Davies, Michael Jonathan; Truscott, R J

2001-01-01

by the protein, or bound chromophore groups, thereby generating excited states (singlet or triplets) or radicals via photo-ionisation. The second major process involves indirect oxidation of the protein via the formation and subsequent reactions of singlet oxygen generated by the transfer of energy to ground...... state (triplet) molecular oxygen by either protein-bound, or other, chromophores. The basic principles behind these mechanisms of photo-oxidation of amino acids, peptides and proteins and the potential selectivity of damage are discussed. Emphasis is placed primarily on the intermediates...

A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma

DEFF Research Database (Denmark)

Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J

2017-01-01

Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues...... in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine...

Oxidative modifications of conjugated and unconjugated linoleic acid during heating.

Science.gov (United States)

Giua, L; Blasi, F; Simonetti, M S; Cossignani, L

2013-10-15

The oxidative stability of conjugated linoleic (CLA) and linoleic (LA) acids in different chemical forms (free acids, methyl esters and homogeneous triacylglycerols) was compared. All model systems were heated at 180°C for different times (15, 30, 45 and 60min). The primary oxidation products were evaluated by spectrophometric analysis, while the volatile compounds were determined by solid phase micro-extraction (SPME), coupled with gas chromatography-mass spectrometry (HRGC-MS). The isomer profile modifications were investigated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) equipped with an UV detector. Generally, peroxide values decreased during the heating time. Among the volatiles, saturated aldehydes were the most represented compounds. Isomerization of cis,trans and trans,cis CLA to trans,trans isomers was observed mainly for the methyl form of CLA. The three different chemical forms of LA never showed isomerization phenomena. Copyright © 2012 Elsevier Ltd. All rights reserved.

Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.

Directory of Open Access Journals (Sweden)

Veronika N Bade

Full Text Available The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls, ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation. ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no

Convergent signaling pathways – interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

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Oxidation of Methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible post-translational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/cells from oxidative stress. However, Met oxidation has been establi...

Protection of naturally occurring antioxidants against oxidative damages to protein

International Nuclear Information System (INIS)

Zhu Hongping; Zhang Zhaoxia; Hao Shumei; Wang Wenfeng; Yao Side

2006-01-01

One of the most compelling theories explaining age-related deterioration is the free radical theory of aging. It has been shown that reactive oxygen species are involved in oxidative damages to biomolecules and this is related to a number of diseases. Proteins, the second most abundant components of cells (next to water by weight), are now increasingly recognized as major biological targets of oxidative damages. Convincing evidences have indicated that damages to protein have been implicated in Alzheimer's disease, Parkinson's disease, cancer, and aging. Antioxidant has been the subject of great attention because they are known to lower the risk of cardiovascular and other diseases. Hydroxycinnamic acid derivatives (HCAs) are antioxidants abundant in tea, red wine, fruits, beverages and various medicinal plants. Results showed that they exhibit remarkable activity for scavenging oxidizing radicals and triplet states. The protective effects of four kinds of HCAs on oxidative damages to lysozyme were investigated in our lab. Protein damages induced by two different paradigms: riboflavin-sensitized photooxidation and hydroxyl ( . OH)-mediated oxidation, were investigated using polyacrylamide gel electrophoresis. HCAs were found to inhibit the cross-linking of protein induced by riboflavin-mediated photooxidation. HCAs also exhibited protection effect on lysozyme damage induced by γ-ray irradiation. The rate constants for quenching triplet state of riboflavin by lysozyme and HCAs were obtained using laser flash photolysis. The protective mechanism was proposed based on the dynamic study. HCAs were found to protect protein against oxidation by scavenging oxidizing species and repairing the damaged protein. (authors)

Covalent modification of cytoskeletal proteins in neuronal cells by tryptamine-4,5-dione

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Yoji Kato

2014-01-01

Full Text Available Serotonin, 5-hydroxytryptamine, is a systemic bioactive amine that acts in the gut and brain. As a substrate of myeloperoxidase in vitro, serotonin is oxidized to tryptamine-4,5-dione (TD, which is highly reactive with thiols. In this work, we successively prepared a monoclonal antibody to quinone-modified proteins and found that the antibody preferentially recognizes the TD–thiol adduct. Using the antibody, we observed that the chloride ion, the predominant physiological substrate for myeloperoxidase in vivo, is not competitive toward the enzyme catalyzed serotonin oxidation process, suggesting that serotonin is a plausible physiological substrate for the enzyme in vivo. Immunocytochemical analyses revealed that TD staining was observed in the cytosol of SH-SY5Y neuroblastoma cells while blot analyses showed that some cellular proteins were preferentially modified. Pull-down analyses confirmed that the cytoskeletal proteins tubulins, vimentin, and neurofilament-L were modified. When pure tubulins were exposed to micromolar levels of synthetic TD, self-polymerization was initially enhanced and then suppressed. These results suggest that serotonin oxidation by myeloperoxidase or the action of other oxidants could cause functional alteration of cellular proteins, which may be related to neurodegeneration processes or irritable bowel syndrome.

Individual whey protein components influence lipid oxidation dependent on pH

DEFF Research Database (Denmark)

Horn, Anna Frisenfeldt; Nielsen, Nina Skall; Jacobsen, Charlotte

In emulsions, lipid oxidation is expected to be initiated at the oil-water interface. The properties of the emulsifier used and the composition at the interface is therefore expected to be of great importance for the resulting oxidation. Previous studies have shown that individual whey protein...... by affecting the preferential adsorption of whey protein components at the interface. The aim of the study was to compare lipid oxidation in 10% fish oil-in-water emulsions prepared with 1% whey protein having either a high concentration of α-lactalbumin, a high concentration of β-lactoglobulin or equal...... amounts of the two. Emulsions were prepared at pH4 and pH7. Emulsions were characterized by their droplet sizes, viscosities, and contents of proteins in the water phase. Lipid oxidation was assessed by PV and secondary volatile oxidation products. Results showed that pH greatly influenced the oxidative...

Biological markers of oxidative stress: Applications to cardiovascular research and practice

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Edwin Ho

2013-01-01

Full Text Available Oxidative stress is a common mediator in pathogenicity of established cardiovascular risk factors. Furthermore, it likely mediates effects of emerging, less well-defined variables that contribute to residual risk not explained by traditional factors. Functional oxidative modifications of cellular proteins, both reversible and irreversible, are a causal step in cellular dysfunction. Identifying markers of oxidative stress has been the focus of many researchers as they have the potential to act as an “integrator” of a multitude of processes that drive cardiovascular pathobiology. One of the major challenges is the accurate quantification of reactive oxygen species with very short half-life. Redox-sensitive proteins with important cellular functions are confined to signalling microdomains in cardiovascular cells and are not readily available for quantification. A popular approach is the measurement of stable by-products modified under conditions of oxidative stress that have entered the circulation. However, these may not accurately reflect redox stress at the cell/tissue level. Many of these modifications are “functionally silent”. Functional significance of the oxidative modifications enhances their validity as a proposed biological marker of cardiovascular disease, and is the strength of the redox cysteine modifications such as glutathionylation. We review selected biomarkers of oxidative stress that show promise in cardiovascular medicine, as well as new methodologies for high-throughput measurement in research and clinical settings. Although associated with disease severity, further studies are required to examine the utility of the most promising oxidative biomarkers to predict prognosis or response to treatment.

Protein O-GlcNAc Modification Increases in White Blood Cells After a Single Bout of Physical Exercise.

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Nagy, Tamás; Kátai, Emese; Fisi, Viktória; Takács, Tamás Tibor; Stréda, Antal; Wittmann, István; Miseta, Attila

2018-01-01

Protein O-linked N -acetylglucosamine (O-GlcNAc) is a dynamic posttranslational modification influencing the function of many intracellular proteins. Recently it was revealed that O-GlcNAc regulation is modified under various stress states, including ischemia and oxidative stress. Aside from a few contradictory studies based on animal models, the effect of exercise on O-GlcNAc is unexplored. To evaluate O-GlcNAc levels in white blood cells (WBC) of human volunteers following physical exercise. Young (age 30 ± 5.2), healthy male volunteers ( n  = 6) were enlisted for the study. Blood parameters including metabolites, ions, "necro"-enzymes, and cell counts were measured before and after a single bout of exercise (2-mile run). From WBC samples, we performed western blots to detect O-GlcNAc modified proteins. The distribution of O-GlcNAc in WBC subpopulations was assessed by flow cytometry. Elevation of serum lactic acid (increased from 1.3 ± 0.4 to 6.9 ± 1.7 mM), creatinine (from 77.5 ± 6.3 U/L to 102.2 ± 7.0 μM), and lactate dehydrogenase (from 318.5 ± 26.2 to 380.5 ± 33.2 U/L) confirmed the effect of exercise. WBC count also significantly increased (from 6.6 ± 1.0 to 8.4 ± 1.4 G/L). The level of O-GlcNAc modified proteins in WBCs showed significant elevation after exercise (85 ± 51%, p  O-GlcNAc status of WBCs. O-GlcNAc modification could be a natural process by which physical activity modulates the immune system. Further research could elucidate the role of O-GlcNAc during exercise and validate O-GlcNAc as a biomarker for fitness assessment.

Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

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Robert M Gill

Full Text Available Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-/hydrogen peroxide (H2O2 release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 μM. This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.

Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

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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.

Carbon Nanotubes Facilitate Oxidation of Cysteine Residues of Proteins.

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Hirano, Atsushi; Kameda, Tomoshi; Wada, Momoyo; Tanaka, Takeshi; Kataura, Hiromichi

2017-10-19

The adsorption of proteins onto nanoparticles such as carbon nanotubes (CNTs) governs the early stages of nanoparticle uptake into biological systems. Previous studies regarding these adsorption processes have primarily focused on the physical interactions between proteins and nanoparticles. In this study, using reduced lysozyme and intact human serum albumin in aqueous solutions, we demonstrated that CNTs interact chemically with proteins. The CNTs induce the oxidation of cysteine residues of the proteins, which is accounted for by charge transfer from the sulfhydryl groups of the cysteine residues to the CNTs. The redox reaction simultaneously suppresses the intermolecular association of proteins via disulfide bonds. These results suggest that CNTs can affect the folding and oxidation degree of proteins in biological systems such as blood and cytosol.

Design and development of anisotropic inorganic/polystyrene nanocomposites by surface modification of zinc oxide nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Han, Xiao [School of Materials Science and Engineering, Tongji University, Shanghai 200092 (China); Research Center for Translational Medicine, East Hospital, the Institute for Biomedical Engineering & Nano Science, Tongji University School of Medicine, Shanghai 200092 (China); Huang, Shiming [Department of Physics, Tongji University, Shanghai 200092 (China); Wang, Yilong, E-mail: yilongwang@tongji.edu.cn [Research Center for Translational Medicine, East Hospital, the Institute for Biomedical Engineering & Nano Science, Tongji University School of Medicine, Shanghai 200092 (China); Shi, Donglu, E-mail: shid@ucmail.uc.edu [Research Center for Translational Medicine, East Hospital, the Institute for Biomedical Engineering & Nano Science, Tongji University School of Medicine, Shanghai 200092 (China); The Materials Science and Engineering Program, College of Engineering and Applied Science, University of Cincinnati, Cincinnati, OH 45221 (United States)

2016-07-01

Anisotropic yolk/shell or Janus inorganic/polystyrene nanocomposites were prepared by combining miniemulsion polymerization and sol–gel reaction. The morphologies of the anisotropic composites were found to be greatly influenced by surface modification of zinc oxide (ZnO) nanoparticle seeds. Two different types of the oleic acid modified ZnO nanoparticles (OA-ZnO) were prepared by post-treatment of commercial ZnO powder and homemade OA-ZnO nanoparticles. The morphologies and properties of the nanocomposites were investigated by transmission electron microscope (TEM), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), and energy dispersive X-ray spectroscopy (EDX). It was found that both post-treated OA-ZnO and in-situ prepared OA-ZnO nanoparticles resulted in the yolk–shell and Janus structure nanocomposites, but with varied size and morphology. These nanocomposites showed stable and strong fluorescence by introducing quantum dots as the co-seeds. The fluorescent anisotropic nanocomposites were decorated separately with surface carboxyl and hydroxyl groups. These composites with unique anisotropic properties will have high potential in biomedical applications, particularly in bio-detection. - Graphical abstract: Design and development of anisotropic inorganic/polystyrene nanocomposites by surface modification of zinc oxide nanoparticles. - Highlights: • Non-magnetic anisotropic yolk/shell or Janus nanocomposites are prepared and characterized. • Different surface modification of zinc oxide (ZnO) nanoparticles results in varied morphology and size of the final product. • Fluorescent anisotropic nanocomposites embodying quantum dots are an ideal candidate for bio-detection applications.

Modification of calcite crystal growth by abalone shell proteins: an atomic force microscope study.

OpenAIRE

Walters, D A; Smith, B L; Belcher, A M; Paloczi, G T; Stucky, G D; Morse, D E; Hansma, P K

1997-01-01

A family of soluble proteins from the shell of Haliotis rufescens was introduced over a growing calcite crystal being scanned in situ by an atomic force microscope (AFM). Atomic step edges on the crystal surface were altered in shape and speed of growth by the proteins. Proteins attached nonuniformly to the surface, indicating different interactions with crystallographically different step edges. The observed changes were consistent with the habit modification induced by this family of protei...

Protein Thiols as an Indication of Oxidative Stress

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Yousef Rezaei Chianeh

2014-06-01

Full Text Available Thiol is an organic compound that contain sulphhydryl group that have a critical role in preventing any involvement of oxidative stress in the cell. These defensive functions are generally considered to be carried out by the low molecular weight thiol glutathione and by cysteine residues in the active sites of proteins such as thioredoxin and peroxiredoxin. In addition, there are thiols exposed on protein surfaces that are not directly involved with protein function, although they can interact with the intracellular environment.The process of protection of the cell against an oxidative damage occur by thiol and cystein residue that has a low molecular weight. These residue are present in the active sites of a protein like, peroxiredoxin and thioredoxin. Apart from intracellular antioxidant defense mechanism by protein thiol, there are presence of thiol in outer surface of protein that are not involved with the function of protein, even though they can interact with intracellular part of the cell. [Archives Medical Review Journal 2014; 23(3.000: 443-456

Evidence for radical-oxidation of plasma proteins in humans

International Nuclear Information System (INIS)

Wang, D.; Davies, M.; Dean, R.; Fu, S.; Taurins, A.; Sullivans, D.

1998-01-01

Oxidation of proteins by radicals has been implicated in many pathological processes. The hydroxyl radical is known to generate protein-bound hydroxylated derivatives of amino acids, for example hydroxyvaline (from Val), hydroxyleucine (from Leu), o-tyrosine (from Phe), and DOPA (from Tyr). In this study, we have investigated the occurrence of these oxidised amino acids in human plasma proteins from both normal subjects and dialysis patients. By employing previously established HPLC methods [Fu et al. Biochemical Journal, 330, 233-239, 1998], we have found that oxidised amino acids exist in normal human plasma proteins (n=32). The level of these oxidised amino acids is not correlated to age. Similar levels of oxidised amino acids are found in the plasma proteins of the dialysis patients (n=6), but a more detailed survey is underway. The relative abundance of the oxidised amino acids is similar to that resulting from oxidation of BSA by hydroxy radicals or Fenton systems [Fu et al. Biochemical Journal, 333, 519-525, 1998]. The results suggest that metal-ion catalysed oxyl-radical chemistry may be a key contributor to the oxidative damage in plasma proteins in vivo in humans

Hormone replacement therapy increases levels of antibodies against heat shock protein 65 and certain species of oxidized low density lipoprotein

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Uint L.

2003-01-01

Full Text Available Hormone replacement therapy (HRT reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp and oxidized low density lipoprotein (LDL have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 ± 0.03 vs 0.558 ± 0.11 and against LDL with a low degree of oxidative modification (0.100 ± 0.01 vs 0.217 ± 0.02 (P<0.05 and P<0.001, respectively, ANOVA. The hormone-mediated immune response may trigger an inflammatory response within the vessel wall and potentially increase plaque burden. Whether or not this immune response is temporary or sustained and deleterious requires further investigation.

Influence of early postmortem protein oxidation on beef quality.

Science.gov (United States)

Rowe, L J; Maddock, K R; Lonergan, S M; Huff-Lonergan, E

2004-03-01

The objective of this study was to examine the effect of early postmortem protein oxidation on the color and tenderness of beef steaks. To obtain a range of oxidation levels, the longissimus lumborum muscles (LM) from both strip loins of 20 steers fed either a finishing diet with vitamin E (1,000 IU per steer daily, minimum of 126 d [VITE]; n = 10 steers) or fed the same finishing diet without vitamin E (CON; n = 10 steers) were used. Within 24 h after slaughter, the LM muscle from each carcass was cut into 2.54-cm-thick steaks and individually vacuum packaged. Steaks from each steer were assigned to a nonirradiated group or an irradiated group. Steaks were irradiated within 26 h postmortem, and were aged at 4 degrees C for 0, 1, 3, 7, and 14 d after irradiation. Steaks from each diet/irradiation/aging time treatment were used to determine color, shear force, and degree of protein oxidation (carbonyl content). Steaks from steers fed the VITE diet had higher (P irradiation, steaks that had been irradiated had lower (P Irradiated steaks, regardless of diet, had lower a* (P irradiated steaks compared to nonirradiated steaks at 0, 1, 3, and 7 d postirradiation. Immunoblot analysis showed that vitamin E supplementation decreased the number and extent of oxidized sarcoplasmic proteins. Protein carbonyl content was positively correlated with Warner-Bratzler shear force values. These results indicate that increased oxidation of muscle proteins early postmortem could have negative effects on fresh meat color and tenderness.

S-Glutathionylation and Redox Protein Signaling in Drug Addiction.

Science.gov (United States)

Womersley, Jacqueline S; Uys, Joachim D

2016-01-01

Drug addiction is a chronic relapsing disorder that comes at a high cost to individuals and society. Therefore understanding the mechanisms by which drugs exert their effects is of prime importance. Drugs of abuse increase the production of reactive oxygen and nitrogen species resulting in oxidative stress. This change in redox homeostasis increases the conjugation of glutathione to protein cysteine residues; a process called S-glutathionylation. Although traditionally regarded as a protective mechanism against irreversible protein oxidation, accumulated evidence suggests a more nuanced role for S-glutathionylation, namely as a mediator in redox-sensitive protein signaling. The reversible modification of protein thiols leading to alteration in function under different physiologic/pathologic conditions provides a mechanism whereby change in redox status can be translated into a functional response. As such, S-glutathionylation represents an understudied means of post-translational protein modification that may be important in the mechanisms underlying drug addiction. This review will discuss the evidence for S-glutathionylation as a redox-sensing mechanism and how this may be involved in the response to drug-induced oxidative stress. The function of S-glutathionylated proteins involved in neurotransmission, dendritic spine structure, and drug-induced behavioral outputs will be reviewed with specific reference to alcohol, cocaine, and heroin. Copyright © 2016. Published by Elsevier Inc.

Effect of Oxidative Damage on the Stability and Dimerization of Superoxide Dismutase 1

OpenAIRE

Petrov, Drazen; Daura, Xavier; Zagrovic, Bojan

2016-01-01

During their life cycle, proteins are subject to different modifications involving reactive oxygen species. Such oxidative damage to proteins may lead to the formation of insoluble aggregates and cytotoxicity and is associated with age-related disorders including neurodegenerative diseases, cancer, and diabetes. Superoxide dismutase 1 (SOD1), a key antioxidant enzyme in human cells, is particularly susceptible to such modifications. Moreover, this homodimeric metalloenzyme has been directly l...

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Proteomic analysis of post-translational modifications

DEFF Research Database (Denmark)

Mann, Matthias; Jensen, Ole N

2003-01-01

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now...... systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel...

The Role of Posttranslational Protein Modifications in Rheumatological Diseases: Focus on Rheumatoid Arthritis

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Andrea Mastrangelo

2015-01-01

Full Text Available The definition of posttranslational modification (PTM encompasses a wide group of chemical reactions that allow modification and modulation of protein functions. The regulation of PTMs is crucial for the activity and survival of the cells. Dysregulation of PTMs has been observed in several pathological conditions, including rheumatoid arthritis (RA. RA is a systemic autoimmune disease primarily targeting the joints. The three PTMs mainly involved in this disease are glycosylation, citrullination, and carbamylation. Glycosylation is essential for antigen processing and presentation and can modulate immunoglobulin activity. Citrullination of self-antigens is strongly associated with RA, as demonstrated by the presence of antibodies directed to anti-citrullinated proteins in patients’ sera. Carbamylation and its dysregulation have been recently associated with RA. Aim of this review is to illustrate the most significant alterations of these PTMs in RA and to evaluate their possible involvement in the pathogenesis of the disease.

Participation of Low Molecular Weight Electron Carriers in Oxidative Protein Folding

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József Mandl

2009-03-01

Full Text Available Oxidative protein folding is mediated by a proteinaceous electron relay system, in which the concerted action of protein disulfide isomerase and Ero1 delivers the electrons from thiol groups to the final acceptor. Oxygen appears to be the final oxidant in aerobic living organisms, although the existence of alternative electron acceptors, e.g. fumarate or nitrate, cannot be excluded. Whilst the protein components of the system are well-known, less attention has been turned to the role of low molecular weight electron carriers in the process. The function of ascorbate, tocopherol and vitamin K has been raised recently. In vitro and in vivo evidence suggests that these redox-active compounds can contribute to the functioning of oxidative folding. This review focuses on the participation of small molecular weight redox compounds in oxidative protein folding.

Increased nitration and carbonylation of proteins in MRL +/+ mice exposed to trichloroethene: Potential role of protein oxidation in autoimmunity

International Nuclear Information System (INIS)

Wang Gangduo; Wang Jianling; Ma Huaxian; Khan, M. Firoze

2009-01-01

Even though reactive oxygen and nitrogen species (RONS) are implicated as mediators of autoimmune diseases (ADs), little is known about contribution of protein oxidation (carbonylation and nitration) in the pathogenesis of such diseases. The focus of this study was, therefore, to establish a link between protein oxidation and induction and/or exacerbation of autoimmunity. To achieve this, female MRL +/+ mice were treated with trichloroethene (TCE), an environmental contaminant known to induce autoimmune response, for 6 or 12 weeks (10 mmol/kg, i.p., every 4 th day). TCE treatment resulted in significantly increased formation of nitrotyrosine (NT) and induction of iNOS in the serum at both 6 and 12 weeks of treatment, but the response was greater at 12 weeks. Likewise, TCE treatment led to greater NT formation, and iNOS protein and mRNA expression in the livers and kidneys. Moreover, TCE treatment also caused significant increases (∼3 fold) in serum protein carbonyls (a marker of protein oxidation) at both 6 and 12 weeks. Significantly increased protein carbonyls were also observed in the livers and kidneys (2.1 and 1.3 fold, respectively) at 6 weeks, and to a greater extent at 12 weeks (3.5 and 2.1 fold, respectively) following TCE treatment. The increases in TCE-induced protein oxidation (carbonylation and nitration) were associated with significant increases in Th1 specific cytokine (IL-2, IFN-γ) release into splenocyte cultures. These results suggest an association between protein oxidation and induction/exacerbation of autoimmune response. The results present a potential mechanism by which oxidatively modified proteins could contribute to TCE-induced autoimmune response and necessitates further investigations for clearly establishing the role of protein oxidation in the pathogenesis of ADs.

Early events in copper-ion catalyzed oxidation of α-synuclein

DEFF Research Database (Denmark)

Tiwari, Manish Kumar; Leinisch, Fabian; Sahin, Cagla

2018-01-01

-synuclein modification using six different molar ratios of Cu2+/H2O2/protein and Cu2+/H2O2/ascorbate/protein resulting in mild to moderate extents of oxidation. For a Cu2+/H2O2/protein molar ratio of 2.3:7.8:1 only low levels of carbonyls were detected (0.078 carbonyls per protein), whereas a molar ratio of 4...

OXIDATIVE MODIFICATION OF PROTEINS AND GLUTATHIONE SYSTEM IN ADIPOCYTES UNDER DIABETES

OpenAIRE

Ye. V. Shakhristova; Ye. A. Stepovaya; V. V. Ivanov; O. L. Nosareva; N. V. Ryazantseva; V. V. Novitsky

2014-01-01

Currently, diabetes ranks third in relation to medical and social significance after cardiovascular diseases and cancer and is the leading cause of blindness; it greatly increases the risk of myocardial infarction, coronary heart disease, nephropathy and hypertension in patients with this disorder; therefore clinical and experimental studies aimed at investigation of diabetes emergence and development mechanisms are urgent.The aim of the study was to investigate the status of oxidative modifi...

Effect of oxidative stress on homer scaffolding proteins.

Directory of Open Access Journals (Sweden)

Igor Nepliouev

Full Text Available Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G. Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.

Advanced oxidation and adsorption modification of dust waste from standard moulding sands

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A. Baliński

2010-04-01

Full Text Available The article discusses the process of advanced oxidation (AO with application of ultrasounds and surface modification of the dust waste collected during dry dedusting of processed moulding sands with bentonite binder. A beneficial effect of both AO and adsorption modification of dust waste, when performed with the selected type of polyelectrolyte, on the technological and mechanical properties of moulding sands prepared with an addition of this dust has been stated. In spite of the bentonite content in moulding sand reduced by 43% and replaced with modified dust waste, the mechanical properties, i.e. the compression and tensile strengths, examined on sand specimens have been improved by 10% and 13%, respectively, with no harm to other basic technological sand properties. At the same time, it was also possible to reduce by about 30% the emission rate of the main gaseous component from the BTEX group, i.e. benzene.

Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction.

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Naudí, Alba; Jové, Mariona; Cacabelos, Daniel; Ayala, Victoria; Cabre, Rosanna; Caro, Pilar; Gomez, José; Portero-Otín, Manuel; Barja, Gustavo; Pamplona, Reinald

2013-02-01

Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N (ε)-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.

Mechanistic understanding of the cysteine capping modifications of antibodies enables selective chemical engineering in live mammalian cells.

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Zhong, Xiaotian; He, Tao; Prashad, Amar S; Wang, Wenge; Cohen, Justin; Ferguson, Darren; Tam, Amy S; Sousa, Eric; Lin, Laura; Tchistiakova, Lioudmila; Gatto, Scott; D'Antona, Aaron; Luan, Yen-Tung; Ma, Weijun; Zollner, Richard; Zhou, Jing; Arve, Bo; Somers, Will; Kriz, Ronald

2017-04-20

Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Surface Modification of Indium Tin Oxide Nanoparticles to Improve Its Distribution in Epoxy-Silica Polymer Matrix

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Mostafa Jafari

2014-10-01

Full Text Available A semiconducting nanoparticle indium tin oxide (ITO was modified with silane groups and for this purpose trimethoxysilane (TMOS precursor was used under specific experimental conditions for surface modification of ITO nanoparticles. It is found that the modification of ITO nanoparticles increases the interactions between the filler and the matrix and subsequently improves the distibution of indium tin oxide nanoparticles in the polymer matrix. The epoxisilica polymer matrix was produced using trimethoxysilane and 3-glycidyloxypropyl trimethoxysilane precursors and ethylenediamine (EDA as curing agent at low temperature by sol-gel process. The sol-gel process was very useful due to its easily controllable process, solution concentration and homogeneity without using expensive and complicated equipments in comparison with other methods. Then, Fourier transform infrared (FTIR spectroscopy was employed to study the formation of Si-O-Si and Si-OH groups on ITO nanoparticles. X-Ray diffraction (XRD technique and thermal gravimetric analysis (TGA were employed to investigate the modification and weight loss of the modified ITO, respectively, as an indication of the presence of organic groups on these nanoparticles. The separation analyzer tests were performed to check the stability of the nanoparticles suspension and it revealed that due to better interaction of nanoparticles with the polymer matrix the stability of modified ITO suspention is higher than the unmodified sample. The morphology and particle distribution were determined by scanning electron microscopy (SEM. It was found that the distibution of modified indium tin oxide in epoxy-silica polymer matrix was improved in comparison with pure ITO.

Biochemistry and pathology of radical-mediated protein oxidation

DEFF Research Database (Denmark)

Dean, R T; Fu, S; Stocker, R

1997-01-01

Radical-mediated damage to proteins may be initiated by electron leakage, metal-ion-dependent reactions and autoxidation of lipids and sugars. The consequent protein oxidation is O2-dependent, and involves several propagating radicals, notably alkoxyl radicals. Its products include several catego...

Structure modification and functionality of whey proteins: quantitative structure-activity relationship approach.

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Nakai, S; Li-Chan, E

1985-10-01

According to the original idea of quantitative structure-activity relationship, electric, hydrophobic, and structural parameters should be taken into consideration for elucidating functionality. Changes in these parameters are reflected in the property of protein solubility upon modification of whey proteins by heating. Although solubility is itself a functional property, it has been utilized to explain other functionalities of proteins. However, better correlations were obtained when hydrophobic parameters of the proteins were used in conjunction with solubility. Various treatments reported in the literature were applied to whey protein concentrate in an attempt to obtain whipping and gelling properties similar to those of egg white. Mapping simplex optimization was used to search for the best results. Improvement in whipping properties by pepsin hydrolysis may have been due to higher protein solubility, and good gelling properties resulting from polyphosphate treatment may have been due to an increase in exposable hydrophobicity. However, the results of angel food cake making were still unsatisfactory.

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor.

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Oesterlin, Lena K; Goody, Roger S; Itzen, Aymelt

2012-04-10

Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.

Oxidative protein modification as predigestive mechanism of the carnivorous plant Dionaea muscipula: an hypothesis based on in vitro experiments.

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Galek, H; Osswald, W F; Elstner, E F

1990-01-01

Aqueous leaf extracts from Dionaea muscipula contain quinones such as the naphthoquinone plumbagin that couple to different NADH-dependent diaphorases, producing superoxide and hydrogen peroxide upon autoxidation. Upon preincubation of Dionaea extracts with certain diaphorases and NADH in the presence of serumalbumin (SA), subsequent tryptic digestion of SA is facilitated. Since the secretroy glands of Droseracea contain proteases and possibly other degradative enzymes it is suggested that the presence of oxygen-activating redox cofactors in the extracts function as extracellular predigestive oxidants which render membrane-bound proteins of the prey (insects) more susceptible to proteolytic attacks.

Protein Topology Determines Cysteine Oxidation Fate: The Case of Sulfenyl Amide Formation among Protein Families

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Defelipe, Lucas A.; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A.; Turjanski, Adrián G.

2015-01-01

Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-Δ12,14-prostaglandin J2

International Nuclear Information System (INIS)

Mishra, Nandita; Kar, Rekha; Singha, Prajjal K.; Venkatachalam, Manjeri A.; McEwen, Donald G.; Saikumar, Pothana

2010-01-01

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 μM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPγS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.

Multiple γ-glutamylation: A novel type of post-translational modification in a diapausing Artemia cyst protein

International Nuclear Information System (INIS)

Hasegawa, Mai; Ikeda, Yuka; Kanzawa, Hideaki; Sakamoto, Mika; Goto, Mina; Tsunasawa, Susumu; Uchiumi, Toshio; Odani, Shoji

2010-01-01

A highly hydrophilic, glutamate-rich protein was identified in the aqueous phenol extract from the cytosolic fraction of brine shrimp (Artemia franciscana) diapausing cysts and termed Artemia phenol soluble protein (PSP). Mass spectrometric analysis revealed the presence of many protein peaks around m/z 11,000, separated by 129 atomic mass units; this value corresponds to that of glutamate, which is strongly suggestive of heterogeneous polyglutamylation. Polyglutamylation has long been known as the functionally important post-translational modification of tubulins, which carry poly(L-glutamic acid) chains of heterogeneous length branching off from the main chain at the γ-carboxy groups of a few specific glutamate residues. In Artemia PSP, however, Edman degradation of enzymatic peptides revealed that at least 13, and presumably 16, glutamate residues were modified by the attachment of a single L-glutamate, representing a hitherto undescribed type of post-translational modification: namely, multiple γ-glutamylation or the addition of a large number of glutamate residues along the polypeptide chain. Although biological significance of PSP and its modification is yet to be established, suppression of in vitro thermal aggregation of lactate dehydrogenase by glutamylated PSP was observed.

Monoaminylation of Fibrinogen and Glia-Derived Proteins: Indication for Similar Mechanisms in Posttranslational Protein Modification in Blood and Brain.

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Hummerich, René; Costina, Victor; Findeisen, Peter; Schloss, Patrick

2015-07-15

Distinct proteins have been demonstrated to be posttranslationally modified by covalent transamidation of serotonin (5-hydropxytryptamin) to glutamine residues of the target proteins. This process is mediated by transglutaminase (TGase) and has been termed "serotonylation." It has also been shown that other biogenic amines, including the neurotransmitters dopamine and norepinephrine, can substitute for serotonin, implying a more general mechanism of "monoaminylation" for this kind of protein modification. Here we transamidated the autofluorescent monoamine monodansylcadaverine (MDC) to purified plasma fibrinogen and to proteins from a primary glia cell culture. Electrophoretic separation of MDC-conjugated proteins followed by mass spectrometry identified three fibrinogen subunits (Aα, Bβ, γ), a homomeric Aα2 dimer, and adducts of >250 kDa molecular weight, as well as several glial proteins. TGase-mediated MDC incorporation was strongly reduced by serotonin, underlining the general mechanism of monoaminylation.

Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications.

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Holtgrefe, Simone; Gohlke, Jochen; Starmann, Julia; Druce, Samantha; Klocke, Susanne; Altmann, Bianca; Wojtera, Joanna; Lindermayr, Christian; Scheibe, Renate

2008-06-01

Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

Oxidative Stress and DNA Methylation in Prostate Cancer

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Krishna Vanaja Donkena

2010-01-01

Full Text Available The protective effects of fruits, vegetables, and other foods on prostate cancer may be due to their antioxidant properties. An imbalance in the oxidative stress/antioxidant status is observed in prostate cancer patients. Genome oxidative damage in prostate cancer patients is associated with higher lipid peroxidation and lower antioxidant levels. Oxygen radicals are associated with different steps of carcinogenesis, including structural DNA damage, epigenetic changes, and protein and lipid alterations. Epigenetics affects genetic regulation, cellular differentiation, embryology, aging, cancer, and other diseases. DNA methylation is perhaps the most extensively studied epigenetic modification, which plays an important role in the regulation of gene expression and chromatin architecture, in association with histone modification and other chromatin-associated proteins. This review will provide a broad overview of the interplay of oxidative stress and DNA methylation, DNA methylation changes in regulation of gene expression, lifestyle changes for prostate cancer prevention, DNA methylation as biomarkers for prostate cancer, methods for detection of methylation, and clinical application of DNA methylation inhibitors for epigenetic therapy.

Oxidized tissue proteins after intestinal reperfusion injury in rats

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Schanaider Alberto

2005-01-01

Full Text Available PURPOSE: To analyse if the carbonyl proteins measurement could be validated as a method that allows the identification of an intestinal oxidative stress after ischemia and reperfusion injury. METHODS: Twenty-five male Wistar rats (n =21 weighting 200 to 250g were divided into three groups. Group I - control (n = 10. Group II - sham (n = 5 and Group III (n = 10 subjected to 60 minutes of intestinal ischemia and equal period of reperfusion. For this purpose it was clamped the superior mesenteric artery in its distal third. Histological changes and carbonyl protein levels were determined in the samples of all groups. In group III, samples of both normal and reperfused ileal segment were studied. RESULTS: All the reperfused segments showed mucosal and submucosal swelling and inflammatory infiltrate of the lamina propria. Levels of carbonyl protein rose in group III, including in the non-ischemic segments. The sensitivity and specificity of the carbonyl protein tissue levels were respectively 94% and 88%. CONCLUSION: The carbonyl protein method is a useful biologic marker of oxidative stress after the phenomenon of intestinal ischemia and reperfusion in rats. It was also noteworthy that the effects of oxidative stress could be seen far from the locus of the primary injury.

Detection and localization of markers of oxidative stress by in situ methods: application in the study of Alzheimer disease.

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Moreira, Paula I; Sayre, Lawrence M; Zhu, Xiongwei; Nunomura, Akihiko; Smith, Mark A; Perry, George

2010-01-01

Oxidative stress is a key factor involved in the development and progression of Alzheimer disease (AD), and it is well documented that free radical oxidative damage, particularly of neuronal lipids, proteins, nucleic acids, and sugars, is extensive in brains of AD patients. The complex chemistry of peroxynitrite has been the subject of intense study and is now evident that there are two principal pathways for protein modification: the first one involves homolytic hydroxyl radical-like chemistry that results in protein-based carbonyls and the second involves electrophilic nitration of vulnerable side chains, in particular the electron-rich aromatic rings of Tyr and Trp. In the presence of buffering bicarbonate, peroxynitrite forms a CO(2) adduct, which augments its reactivity. Formation of 3-nitrotyrosine by this route has become the classical protein marker specifically for the presence of peroxynitrite. Protein-based carbonyls can be detected by two methods: (i) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and detection of the protein-bound hydrazones using an enzyme-linked anti-2,4-dinitrophenyl antibody and (ii) derivatization with biotin-hydrazide and detection of the protein-bound acyl hydrazone with enzyme-linked avidin or streptavidin. Glycation of proteins by reducing sugars (Maillard reaction) results in a profile of time-dependent adduct evolution rendering susceptibility to oxidative elaboration. In addition, oxidative stress can result in oxidized sugar derivatives which can subsequently modify protein through a process known as glycoxidation. Of more general importance, oxidative stress results in lipid peroxidation and the production of a range of electrophilic and mostly bifunctional aldehydes that modify numerous proteins. The more important protein modifications are referred to as advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). Protein modification can result in both non-cross-link and cross-link AGEs

Mechanisms of protein misfolding: Novel therapeutic approaches to protein-misfolding diseases

DEFF Research Database (Denmark)

Salahuddin, Parveen; Siddiqi, Mohammad Khursheed; Khan, Sanaullah

2016-01-01

’s disease (PD), Alzheimer’s disease (AD), Prion disease and Amylo lateral Sclerosis (ALS). Furthermore, tau protein shows intrinsically disorder conformation; therefore its interaction with microtubule is impaired and this protein undergoes aggregation. This is also underlying cause of Alzheimers and other......In protein misfolding, protein molecule acquires wrong tertiary structure, thereby induces protein misfolding diseases. Protein misfolding can occur through various mechanisms. For instance, changes in environmental conditions, oxidative stress, dominant negative mutations, error in post......-translational modifications, increase in degradation rate and trafficking error. All of these factors cause protein misfolding thereby leading to diseases conditions. Both in vitro and in vivo observations suggest that partially unfolded or misfolded intermediates are particularly prone to aggregation. These partially...

Mass Spectrometry of Intact Proteins Reveals +98 u Chemical Artifacts Following Precipitation in Acetone.

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Güray, Melda Z; Zheng, Shi; Doucette, Alan A

2017-02-03

Protein precipitation in acetone is frequently employed ahead of mass spectrometry for sample preconcentration and purification. Unfortunately, acetone is not chemically inert; mass artifacts have previously been observed on glycine-containing peptides when exposed to acetone under acidic conditions. We herein report a distinct chemical modification occurring at the level of intact proteins when incubated in acetone. This artifact manifests as one or more satellite peaks in the MS spectrum of intact protein, spaced 98 u above the mass of the unmodified protein. Other artifacts (+84, +112 u) also appear upon incubation of proteins or peptides in acetone. The reaction is pH-sensitive, being suppressed when proteins are exposed to acetone under acidic conditions. The +98 u artifact is speculated to originate through an intermediate product of aldol condensation of acetone to form diacetone alcohol and mesityl oxide. A +98 u product could originate from nucleophilic attack on mesityl oxide or through condensation with diacetone alcohol. Given the extent of modification possible upon exposure of proteins to acetone, particularly following overnight solvent exposure or incubation at room temperature, an awareness of the variables influencing this novel modification is valued by proteomics researchers who employ acetone precipitation for protein purification.

MWW-type titanosilicate synthesis, structural modification and catalytic applications to green oxidations

CERN Document Server

Wu, Peng; Xu, Le; Liu, Yueming; He, Mingyuan

2013-01-01

This book provides a comprehensive review of a new generation of selective oxidation titanosilicate catalysts with the MWW topology (Ti-MWW) based on the research achievements of the past 12 years. It gives an overview of the synthesis, structure modification and catalytic properties of Ti-MWW. Ti-MWW can readily be prepared by means of direct hydrothermal synthesis with crystallization-supporting agents, using dual-structure-directing agents and a dry-gel conversion technique. It also can be post-synthesized through unique reversible structure transformation and liquid-phase isomorphous subst

Modification of the protein corona–nanoparticle complex by physiological factors

Energy Technology Data Exchange (ETDEWEB)

Braun, Nicholas J.; DeBrosse, Madeleine C.; Hussain, Saber M. [Molecular Bioeffects Branch, Bioeffects Division, Human Effectiveness Directorate, 711 Human Performance Wing, Air Force Research Laboratory, Wright Patterson AFB, 2729 R. St, Bldg 837, Dayton, OH, 45433 (United States); Comfort, Kristen K., E-mail: kcomfort1@udayton.edu [Department of Chemical and Materials Engineering, University of Dayton, 524 Kettering Laboratories, 300 College Park, Dayton, OH 45469 (United States)

2016-07-01

Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona–NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13 nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications. - Highlights: • Dynamic flow increased the size of the gold nanoparticle protein corona. • Exposure to biological fluids altered protein corona size and composition. • Interstitial fluid modified the nano-cellular interface and deposition efficiency. • Tannic acid coated nanoparticles induced toxicity in an interstitial environment.

Modification of the protein corona–nanoparticle complex by physiological factors

International Nuclear Information System (INIS)

Braun, Nicholas J.; DeBrosse, Madeleine C.; Hussain, Saber M.; Comfort, Kristen K.

2016-01-01

Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona–NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13 nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications. - Highlights: • Dynamic flow increased the size of the gold nanoparticle protein corona. • Exposure to biological fluids altered protein corona size and composition. • Interstitial fluid modified the nano-cellular interface and deposition efficiency. • Tannic acid coated nanoparticles induced toxicity in an interstitial environment.

Self-assembling triblock proteins for biofunctional surface modification

Science.gov (United States)

Fischer, Stephen E.

of the triblock protein hydrogels, and the ease of introducing multiple functionalities to a substrate surface, a surface coating is tailored for neural stem cell culture in order to improve proliferation on the scaffold, while maintaining the stem cell phenotype. These studies demonstrate the unique advantages of genetic engineering over traditional techniques for surface modification. In addition to their unmatched sequence fidelity, recombinant proteins can easily be modified with bioactive ligands and their organization into coherent, supramolecular structures mimics natural self-assembly processes.

Modification of oxide films by ion implantation: TiO2-films modified by Ti+ and O+ as example

International Nuclear Information System (INIS)

Schultze, J.W.; Elfenthal, L.; Leitner, K.; Meyer, O.

1988-01-01

Oxide films can be modified by ion implantation. Changes in the electrochemical properties of the films are due to the deposition profile of the implanted ion, ie doping and stoichiometric changes, as well as to the radiation damage. The latter is due to the formation of Frenkel defects and at high concentrations to a complete amorphization of the oxide film. TiOsub(x)-films with 1 + - and O + -ions into anodic oxide films on titanium. The electrode capacity shows always the behaviour of an n-type semiconductor with an almost constant flatband potential but a strong maximum donor concentration at about 3% Ti + concentration. Oxygen implantation, on the other hand, causes a small increase of donor concentration only at high concentration of O + . Electron transfer reactions show strong modifications of the electronic behaviour of the oxide film with a maximum again at 3% titanium. Photocurrent spectra prove the increasing amorphization and show interband states 2.6 eV above the VB or below the CB. During repassivation measurements at various potentials different defects formed by Ti + - and O + -implantation become mobile. A tentative model of the band structure is constructed which takes into account the interband states due to localised Ti + - and O + -ions. The modification of ion implanted oxide films is compared with the effects of other preparation techniques. (author)

6-Hydroxydopamine Inhibits the Hepatitis C Virus through Alkylation of Host and Viral Proteins and the Induction of Oxidative Stress.

Science.gov (United States)

Lafreniere, Matthew A; Powdrill, Megan H; Singaravelu, Ragunath; Pezacki, John Paul

2016-11-11

Many viruses, including the hepatitis C virus (HCV), are dependent on the host RNA silencing pathway for replication. In this study, we screened small molecule probes, previously reported to disrupt loading of the RNA-induced silencing complex (RISC), including 6-hydroxydopamine (6-OHDA), suramin (SUR), and aurintricarboxylic acid (ATA), to examine their effects on viral replication. We found that 6-OHDA inhibited HCV replication; however, 6-OHDA was a less potent inhibitor of RISC than either SUR or ATA. By generating a novel chemical probe (6-OHDA-yne), we determined that 6-OHDA covalently modifies host and virus proteins. Moreover, 6-OHDA was shown to be an alkylating agent that is capable of generating adducts with a number of enzymes involved in the oxidative stress response. Furthermore, modification of viral enzymes with 6-OHDA and 6-OHDA-yne was found to inhibit their enzymatic activity. Our findings suggest that 6-OHDA is a probe for oxidative stress as well as protein alkylation, and these properties together contribute to the antiviral effects of this compound.

A central role for ubiquitination within a circadian clock protein modification code

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Katarina eStojkovic

2014-08-01

Full Text Available Circadian rhythms, endogenous cycles of about 24 h in physiology, are generated by a master clock located in the suprachiasmatic nucleus of the hypothalamus and other clocks located in the brain and peripheral tissues. Circadian disruption is known to increase the incidence of various illnesses, such as mental disorders, metabolic syndrome and cancer. At the molecular level, periodicity is established by a set of clock genes via autoregulatory translation-transcription feedback loops. This clock mechanism is regulated by post-translational modifications such as phosphorylation and ubiquitination, which set the pace of the clock. Ubiquitination in particular has been found to regulate the stability of core clock components, but also other clock protein functions. Mutation of genes encoding ubiquitin ligases can cause either elongation or shortening of the endogenous circadian period. Recent research has also started to uncover roles for deubiquitination in the molecular clockwork. Here we review the role of the ubiquitin pathway in regulating the circadian clock and we propose that ubiquitination is a key element in a clock protein modification code that orchestrates clock mechanisms and circadian behavior over the daily cycle.

Modification of liposomes with proteins by dansyl-labeled heterobifunctional crosslinker.

Science.gov (United States)

Chen, Tao; Wang, Rutao; Lu, Tingting; Liang, Guozheng; Lu, Tingli

2011-07-01

The introduction of a fluorescent chromaphore into bifunctional crosslinkers results in a molecule with normal crosslinker properties and a fluorescent group for straightforward quantification. This work describes the synthesis of the dansyl-labeled heterobifunctional crosslinker N-succinimidyl ε-N-dansyl α-N-(acetylthio)acetyllysine (dansyl-ATA-lysine-NHS) containing reactive N-hydroxysuccinimidyl (NHS) ester and sulfhydryl groups. The application of this crosslinker to conjugation of bovine serum albumin (BSA) protein to the surface of a liposome containing maleimide functions is also demonstrated. BSA was modified with the dansyl-labeled crosslinker and subsequently conjugated to liposomes containing reactive phospholipid derivative N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine and the degree of modification and conjugation were quantitatively determined by measuring the fluorescence emission of the dansyl group. The reliability of the fluorescence quantification was confirmed by a micro bio-barcode assay protein assay.

Beta-actin deficiency with oxidative posttranslational modifications in Rett syndrome erythrocytes: insights into an altered cytoskeletal organization.

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Alessio Cortelazzo

Full Text Available Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenance of cell shape to transcription regulation, was evaluated in the erythrocyte membranes from patients with typical Rett syndrome (RTT and methyl CpG binding protein 2 (MECP2 gene mutations. RTT, affecting almost exclusively females with an average frequency of 1∶10,000 female live births, is considered the second commonest cause of severe cognitive impairment in the female gender. Evaluation of beta-actin was carried out in a comparative cohort study on red blood cells (RBCs, drawn from healthy control subjects and RTT patients using mass spectrometry-based quantitative analysis. We observed a decreased expression of the beta-actin isoforms (relative fold changes for spots 1, 2 and 3: -1.82±0.15, -2.15±0.06, and -2.59±0.48, respectively in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, beta-actin from RTT patients also showed a dramatic increase in oxidative posttranslational modifications (PTMs as the result of its binding with the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE. Our findings demonstrate, for the first time, a beta-actin down-regulation and oxidative PTMs for RBCs of RTT patients, thus indicating an altered cytoskeletal organization.

Erectile dysfunction and diabetes: Association with the impairment of lipid metabolism and oxidative stress.

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Belba, Arben; Cortelazzo, Alessio; Andrea, Giansanti; Durante, Jacopo; Nigi, Laura; Dotta, Francesco; Timperio, Anna Maria; Zolla, Lello; Leoncini, Roberto; Guerranti, Roberto; Ponchietti, Roberto

2016-01-01

To test the hypothesis that exists an association of non-diabetic and diabetic patients suffering from erectile dysfunction (ED) with lipid metabolism and oxidative stress. Clinical and laboratory characteristics in non-diabetic (n = 30, middle age range: 41–55.5 years; n = 25, old age range: 55.5–73), diabetic ED patients (n = 30, age range: 55.5–75 years) and diabetic patients (n = 25, age range: 56–73.25), were investigated. Proteomic analysis was performed to identify differentially expressed plasma proteins and to evaluate their oxidative posttranslational modifications. A decreased level of high-density lipoproteins in all ED patients (P < 0.001, C.I. 0.046–0.10), was detected by routine laboratory tests. Proteomic analysis showed a significant decreased expression (P < 0.05) of 5 apolipoproteins (i.e. apolipoprotein H, apolipoprotein A4, apolipoprotein J, apolipoprotein E and apolipoprotein A1) and zinc-alpha-2-glycoprotein, 50% of which are more oxidized proteins. Exclusively for diabetic ED patients, oxidative posttranslational modifications for prealbumin, serum albumin, serum transferrin and haptoglobin markedly increased. Showing evidence for decreased expression of apolipoproteins in ED and the remarkable enhancement of oxidative posttranslational modifications in diabetes-associated ED, considering type 2 diabetes mellitus and age as independent risk factors involved in the ED pathogenesis, lipid metabolism and oxidative stress appear to exert a complex interplay in the disease.

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

International Nuclear Information System (INIS)

Hegele, Joerg; Buetler, Timo; Delatour, Thierry

2008-01-01

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N ε -fructoselysine (FL), N ε -carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 ± 3.81 nmol CML per μmol of free Lys (Lys free ) and 81.5 ± 87.8 nmol Pyr μmol -1 Lys free -1 vs. 3.72 ± 1.29 nmol FL μmol -1 Lys free -1 . In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 ± 0.08 nmol FL μmol -1 of protein-bound Lys (Lys p-b ), 0.04 ± 0.03 nmol CML μmol -1 Lys p-b -1 and 0.06 ± 0.02 nmol Pyr μmol -1 Lys p-b -1 . It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products

Analysis and Chemistry of Novel Protein Oxidation Markers in Vivo.

Science.gov (United States)

Henning, Christian; Liehr, Kristin; Girndt, Matthias; Ulrich, Christof; Glomb, Marcus A

2018-05-09

Proteins continually undergo spontaneous oxidation reactions, which lead to changes in structure and function. The quantitative assessment of protein oxidation adducts provides information on the level of exposure to reactive precursor compounds with a high oxidizing potential and reactive oxygen species (ROS). In the present work, we introduce N 6 -(2-hydroxyethyl)lysine as a novel marker based on the ratio of glycolaldehyde and its oxidized form glyoxal. The high analytical potential was proven with a first set of patients undergoing hemodialysis versus healthy controls, in comparison with well-established parameters for oxidative stress. In vitro experiments with N 1 - t-BOC-lysine and N 1 - t-BOC-arginine enlightened the mechanistic relationship of glycolaldehyde and glyoxal. Oxidation was strongly dependent on the catalytic action of the ε-amino moiety of lysine. Investigations on the formation of N 6 -carboxymethyl lysine revealed glycolaldehyde-imine as the more reactive precursor, even though an additional oxidative step is required. As a result, a novel and very effective alternative mechanism was unraveled.

Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae.

Science.gov (United States)

Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè

2016-03-16

Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein Tyrosine Nitration: Biochemical Mechanisms and Structural Basis of its Functional Effects

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Radi, Rafael

2012-01-01

CONSPECTUS The nitration of protein tyrosine residues to 3-nitrotyrosine represents an oxidative postranslational modification that unveils the disruption of nitric oxide (•NO) signaling and metabolism towards pro-oxidant processes. Indeed, excess levels of reactive oxygen species in the presence of •NO or •NO-derived metabolites lead to the formation of nitrating species such as peroxynitrite. Thus, protein 3-nitrotyrosine has been established as a biomarker of cell, tissue and systemic “nitroxidative stress”. Moreover, tyrosine nitration modifies key properties of the amino acid (i.e. phenol group pKa, redox potential, hydrophobicity and volume). Thus, the incorporation of a nitro group (−NO2) to protein tyrosines can lead to profound structural and functional changes, some of which contribute to altered cell and tissue homeostasis. In this Account, I describe our current efforts to define 1) biologically-relevant mechanisms of protein tyrosine nitration and 2) how this modification can cause changes in protein structure and function at the molecular level. First, the relevance of protein tyrosine nitration via free radical-mediated reactions (in both peroxynitrite-dependent or independent pathways) involving the intermediacy of tyrosyl radical (Tyr•) will be underscored. This feature of the nitration process becomes critical as Tyr• can take variable fates, including the formation of 3-nitrotyrosine. Fast kinetic techniques, electron paramagnetic resonance (EPR) studies, bioanalytical methods and kinetic simulations have altogether assisted to characterize and fingerprint the reactions of tyrosine with peroxynitrite and one-electron oxidants and its further evolution to 3-nitrotyrosine. Recent findings show that nitration of tyrosines in proteins associated to biomembranes is linked to the lipid peroxidation process via a connecting reaction that involves the one-electron oxidation of tyrosine by lipid peroxyl radicals (LOO•). Second

Volatile profile, lipid oxidation and protein oxidation of irradiated ready-to-eat cured turkey meat products

International Nuclear Information System (INIS)

Feng, Xi; Ahn, Dong Uk

2016-01-01

Irradiation had little effects on the thiobarbituric acid reactive substances (TBARS) values in ready-to-eat (RTE) turkey meat products, while it increased protein oxidation at 4.5 kGy. The volatile profile analyses indicated that the amount of sulfur compounds increased linearly as doses increased in RTE turkey meat products. By correlation analysis, a positive correlation was found between benzene/ benzene derivatives and alcohols with lipid oxidation, while aldehydes, ketones and alkane, alkenes and alkynes were positively correlated with protein oxidation. Principle component analysis showed that irradiated meat samples can be discriminated by two categories of volatile compounds: Strecker degradation products and radiolytic degradation products. The cluster analysis of volatile data demonstrated that low-dose irradiation had minor effects on the volatile profile of turkey sausages (<1.5 kGy). However, as the doses increased, the differences between the irradiated and non-irradiated cured turkey products became significant. - Highlights: • Irradiation had little effects on lipid oxidation of ready-to-eat cured turkey. • 4.5 kGy irradiation increased protein oxidation. • Irradiated samples were isolated due to Strecker/radiolytic degradation products. • 1.5 kGy irradiation had limited effects on the volatile profile of turkey sausages. • Dimethyl disulfide can be used as a potential marker for irradiated meat products.

Thiol oxidation of hemolymph proteins in oysters Crassostrea brasiliana as markers of oxidative damage induced by urban sewage exposure.

Science.gov (United States)

Trevisan, Rafael; Flores-Nunes, Fabrício; Dolores, Euler S; Mattos, Jacó J; Piazza, Clei E; Sasaki, Sílvio T; Taniguchi, Satie; Montone, Rosalinda C; Bícego, Márcia C; Dos Reis, Isis M M; Zacchi, Flávia L; Othero, Bárbara N M; Bastolla, Camila L V; Mello, Danielle F; Fraga, Ana Paula M; Wendt, Nestor; Toledo-Silva, Guilherme; Razzera, Guilherme; Dafre, Alcir L; de Melo, Cláudio M R; Bianchini, Adalto; Marques, Maria R F; Bainy, Afonso C D

2017-07-01

Urban sewage is a concerning issue worldwide, threatening both wildlife and human health. The present study investigated protein oxidation in mangrove oysters (Crassostrea brasiliana) exposed to seawater from Balneário Camboriú, an important tourist destination in Brazil that is affected by urban sewage. Oysters were exposed for 24 h to seawater collected close to the Camboriú River (CAM1) or 1 km away (CAM2). Seawater from an aquaculture laboratory was used as a reference. Local sewage input was marked by higher levels of coliforms, nitrogen, and phosphorus in seawater, as well as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), linear alkylbenzenes (LABs), and fecal steroid in sediments at CAM1. Exposure of oysters to CAM1 caused marked bioaccumulation of LABs and decreased PAH and PCB concentrations after exposure to both CAM1 and CAM2. Protein thiol oxidation in gills, digestive gland, and hemolymph was evaluated. Lower levels of reduced protein thiols were detected in hemolymph from CAM1, and actin, segon, and dominin were identified as targets of protein thiol oxidation. Dominin susceptibility to oxidation was confirmed in vitro by exposure to peroxides and hypochlorous acid, and 2 cysteine residues were identified as potential sites of oxidation. Overall, these data indicate that urban sewage contamination in local waters has a toxic potential and that protein thiol oxidation in hemolymph could be a useful biomarker of oxidative stress in bivalves exposed to contaminants. Environ Toxicol Chem 2017;36:1833-1845. © 2016 SETAC. © 2016 SETAC.

The structure of the hypothetical protein smu.1377c from Streptococcus mutans suggests a role in tRNA modification

International Nuclear Information System (INIS)

Fu, Tian-Min; Liu, Xiang; Li, Lanfen; Su, Xiao-Dong

2010-01-01

The crystal structure of smu.1377c, a hypothetical protein from S. mutans, shows a similar fold to Sua5-YciO-YrdC-family proteins and indicates its functional role in tRNA modification. Members of the Sua5-YciO-YrdC protein family are found in both eukaryotes and prokaryotes and possess a conserved α/β twisted open-sheet fold. The Escherichia coli protein YrdC has been shown to be involved in modification of tRNA. The crystal structure of smu.1377c, a hypothetical protein from Streptococcus mutans, has been determined to 2.25 Å resolution. From structure analysis and comparison, it is shown that smu.1377c is a member of the Sua5-YciO-YrdC family and that it may play the same role as E. coli YrdC

Isolation of low density lipoprotein (LDL with its modification by Copper ion and Malondialdehyde (MDA

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Doosty M

1999-06-01

Full Text Available Oxidation of low density lipoproteins (LDLs is belived to be an important step in the pathogenesis of atherosclerosis. During oxidation, LDL particle undergoes a large number of structural changes that alters its biological properties, so it becomes atherogenic. To study atherogenic proteins, usually two forms of modified LDLs, including Cu2+-oxidized LDL (ox-LDL and malondialdehyde (MDA modified LDL (mal-LDL are used. In this study, LDL was isolated from 72 ml freshly prepared plasma by sequential Floatation Ultracentrifugation (SFU, which resulted in separation of 12.5 mg LDL protein. LDL oxidation was accomplished in Phosphate Buffered Saline (PBS with 2µM cupric sulfate, and mal-LDL was prepared by incubating LDL in PBS with 0.5 M solution of freshly prepared MDA. These modifications were evaluated by measuring optical density at 234 nm, Thiobarbitoric Acid Reactive Substances (TBARS, and electrophoretic mobility at pH 8.6. The increase of 234 nm absorption reflected initiation of LDL oxidation. TBARS of ox-LDL and mal-LDL was 80 Nm MAD/mg LDL protein and 400 nm MDA/mg LDL protein, respectively. Electrophoretic mobility of ox-LDL and mal-LDL, in respect to native LDL (n-LDL, were increased.

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

Science.gov (United States)

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.

Proteomic detection of oxidized and reduced thiol proteins in cultured cells.

Science.gov (United States)

Cuddihy, Sarah L; Baty, James W; Brown, Kristin K; Winterbourn, Christine C; Hampton, Mark B

2009-01-01

The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

Autoantibodies to Posttranslational Modifications in Rheumatoid Arthritis

Science.gov (United States)

Burska, Agata N.; Hunt, Laura; Strollo, Rocky; Ryan, Brent J.; Vital, Ed; Nissim, Ahuva; Winyard, Paul G.; Emery, Paul; Ponchel, Frederique

2014-01-01

Autoantibodies have been associated with human pathologies for a long time, particularly with autoimmune diseases (AIDs). Rheumatoid factor (RF) is known since the late 1930s to be associated with rheumatoid arthritis (RA). The discovery of anticitrullinated protein antibodies in the last century has changed this and other posttranslational modifications (PTM) relevant to RA have since been described. Such PTM introduce neoepitopes in proteins that can generate novel autoantibody specificities. The recent recognition of these novel specificities in RA provides a unique opportunity to understand human B-cell development in vivo. In this paper, we will review the three of the main classes of PTMs already associated with RA: citrullination, carbamylation, and oxidation. With the advancement of research methodologies it should be expected that other autoantibodies against PTM proteins could be discovered in patients with autoimmune diseases. Many of such autoantibodies may provide significant biomarker potential. PMID:24782594

PARAQUAT TOLERANCE3 Is an E3 Ligase That Switches off Activated Oxidative Response by Targeting Histone-Modifying PROTEIN METHYLTRANSFERASE4b.

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Chao Luo

2016-09-01

Full Text Available Oxidative stress is unavoidable for aerobic organisms. When abiotic and biotic stresses are encountered, oxidative damage could occur in cells. To avoid this damage, defense mechanisms must be timely and efficiently modulated. While the response to oxidative stress has been extensively studied in plants, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3 as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ubiquitin ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b upregulates the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. On the other hand, PRMT4b is recognized by PQT3 for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress and found that histone modification by PRMT4b at APX1 and GPX1 loci plays an important role in oxidative stress tolerance.

Hypochlorite-induced oxidation of amino acids, peptides and proteins

DEFF Research Database (Denmark)

Hawkins, C L; Pattison, D I; Davies, Michael Jonathan

2003-01-01

Activated phagocytes generate the potent oxidant hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is known to react with a number of biological targets including proteins, DNA, lipids and cholesterol. Proteins are likely to be major targets for reactio...

The effect of long-term treatment with coenzyme Q10 on nucleic acid modifications by oxidation in children with Down syndrome

DEFF Research Database (Denmark)

Larsen, Emil List; Padella, Lucia; Bergholdt, Helle Kirstine Mørup

2018-01-01

Elevated levels of oxidative nucleic acid modifications have been proposed to be associated with some of the clinical characteristics of Down syndrome. Oral intake of coenzyme Q10 improves oxidative status and shows a tendency toward protective effect on DNA oxidation in certain age groups...... of children with Down syndrome. Here, we demonstrate that long-term (i.e., 4 years) treatment with coenzyme Q10 (ubiquinone) at the dosage of 4 mg/kg/d does not affect whole body DNA and RNA oxidation....

Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

OpenAIRE

Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

2015-01-01

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we sh...

Genetic Variation and Its Reflection on Posttranslational Modifications in Frequency Clock and Mating Type a-1 Proteins in Sordaria fimicola

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Rabia Arif

2017-01-01

Full Text Available Posttranslational modifications (PTMs occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment on proteins or various amino acids pickings and their ultimate impact on consensus sequences present in mating type proteins using bioinformatics tools. Furthermore, we have also measured and walked through genomic DNA of various Sordaria strains to determine genetic diversity by genotyping the short sequence repeats (SSRs of wild strains of S. fimicola collected from contrasting environments of two opposing slopes (harsh and xeric south facing slope and mild north facing slope of Evolution Canyon (EC, Israel. Based on the whole genome sequence of S. macrospora, we targeted 20 genomic regions in S. fimicola which contain short sequence repeats (SSRs. Our data revealed genetic variations in strains from south facing slope and these findings assist in the hypothesis that genetic variations caused by stressful environments lead to evolution.

Genetic Variation and Its Reflection on Posttranslational Modifications in Frequency Clock and Mating Type a-1 Proteins in Sordaria fimicola.

Science.gov (United States)

Arif, Rabia; Akram, Faiza; Jamil, Tazeen; Mukhtar, Hamid; Lee, Siu Fai; Saleem, Muhammad

2017-01-01

Posttranslational modifications (PTMs) occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment on proteins or various amino acids pickings and their ultimate impact on consensus sequences present in mating type proteins using bioinformatics tools. Furthermore, we have also measured and walked through genomic DNA of various Sordaria strains to determine genetic diversity by genotyping the short sequence repeats (SSRs) of wild strains of S. fimicola collected from contrasting environments of two opposing slopes (harsh and xeric south facing slope and mild north facing slope) of Evolution Canyon (EC), Israel. Based on the whole genome sequence of S. macrospora , we targeted 20 genomic regions in S. fimicola which contain short sequence repeats (SSRs). Our data revealed genetic variations in strains from south facing slope and these findings assist in the hypothesis that genetic variations caused by stressful environments lead to evolution.

Surface modification of promising cerium oxide nanoparticles for nanomedicine applications

KAUST Repository

Nanda, Himansu Sekhar

2016-11-14

Cerium oxide nanoparticles (CNPs) or nanoceria have emerged as a potential nanomedicine for the treatment of several diseases such as cancer. CNPs have a natural tendency to aggregate or agglomerate in their bare state, which leads to sedimentation in a biological environment. Since the natural biological environment is essentially aqueous, nanoparticle surface modification using suitable biocompatible hydrophilic chemical moieties is highly desirable to create effective aqueous dispersions. In this report, (6-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-hexyl)triethoxysilane was used as a functional, biocompatible organosilane to modify the surface of CNPs to produce promising nanoparticles which open substantial therapeutic avenues. The surface modified nanoparticles were produced in situ via an ammonia-induced ethylene glycol-assisted precipitation method and were characterized using complimentary characterization techniques. The interaction between the functional moiety and the nanoparticle was studied using powerful cross polarization/magic angle sample spinning solid state nuclear magnetic resonance spectroscopy. The surface-modified nanoparticles were extremely small and demonstrated a significant improvement in aqueous dispersibility. Moreover, the existence of a strong ionic coordination between the functional moiety and the surface of the nanoparticle was realised, indicating that the surface modified nanoceria are stable and that the nanoparticles should demonstrate an enhanced circulation time in a biological environment. The surface modification approach should be promising for the production of CNPs for nanomedicine applications. © The Royal Society of Chemistry.

Accelerated differentiation of osteoblast cells on polycaprolactone scaffolds driven by a combined effect of protein coating and plasma modification

Energy Technology Data Exchange (ETDEWEB)

Yildirim, Eda D; Gueceri, Selcuk; Sun, Wei [Department of Mechanical Engineering and Mechanics, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Besunder, Robyn; Allen, Fred [Drexel University, School of Biomedical Engineering Science and Health System, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Pappas, Daphne, E-mail: edy22@drexel.ed [Army Research Laboratory, Aberdeen Proving Ground, MD 21005 (United States)

2010-03-15

A combined effect of protein coating and plasma modification on the quality of the osteoblast-scaffold interaction was investigated. Three-dimensional polycaprolactone (PCL) scaffolds were manufactured by the precision extrusion deposition (PED) system. The structural, physical, chemical and biological cues were introduced to the surface through providing 3D structure, coating with adhesive protein fibronectin and modifying the surface with oxygen-based plasma. The changes in the surface properties of PCL after those modifications were examined by contact angle goniometry, surface energy calculation, surface chemistry analysis (XPS) and surface topography measurements (AFM). The effects of modification techniques on osteoblast short-term and long-term functions were examined by cell adhesion, proliferation assays and differentiation markers, namely alkaline phosphatase activity (ALP) and osteocalcin secretion. The results suggested that the physical and chemical cues introduced by plasma modification might be sufficient for improved cell adhesion, but for accelerated osteoblast differentiation the synergetic effects of structural, physical, chemical and biological cues should be introduced to the PCL surface.

3-Hydroxylysine, a potential marker for studying radical-induced protein oxidation

DEFF Research Database (Denmark)

Morin, B; Bubb, W A; Davies, Michael Jonathan

1998-01-01

albumin (BSA) and human low-density lipoprotein (LDL)] and diseased human tissues (atherosclerotic plaques and lens cataractous proteins). This work was aimed at investigating oxidized lysine as a sensitive marker for protein oxidation, as such residues are present on protein surfaces, and are therefore...... likely to be particularly susceptible to oxidation by radicals in bulk solution. HO* attack on lysine in the presence of oxygen, followed by NaBH4 reduction, is shown to give rise to (2S)-3-hydroxylysine [(2S)-2,6-diamino-3-hydroxyhexanoic acid], (2S)-4-hydroxylysine [(2S)-2,6-diamino-4-hydroxyhexanoic...... acid], (2S, 5R)-5-hydroxylysine [(2S,5R)-2,6-diamino-5-hydroxyhexanoic acid], and (2S,5S)-5-hydroxylysine [(2S,5S)-2,6-diamino-5-hydroxyhexanoic acid]. 5-Hydroxylysines are natural products formed by lysyl oxidase and are therefore not good markers of radical-mediated oxidation. The other...

Reduced protein oxidation in Wistar rats supplemented with marine ω3 PUFAs.

Science.gov (United States)

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 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.

Stable markers of oxidant damage to proteins and their application in the study of human disease

DEFF Research Database (Denmark)

Davies, Michael Jonathan; Fu, S; Wang, H

1999-01-01

The mechanisms of formation and the nature of the altered amino acid side chains formed on proteins subjected to oxidant attack are reviewed. The use of stable products of protein side chain oxidation as potential markers for assessing oxidative damage in vivo in humans is discussed. The methods...... developed in the authors laboratories are outlined, and the advantages and disadvantages of these techniques compared with other methodologies for assessing oxidative damage to proteins and other macromolecules. Evidence is presented to show that protein oxidation products are sensitive markers of oxidative...... damage, that the pattern of products detected may yield information as to the nature of the original oxidative insult, and that the levels of oxidized side-chains can, in certain circumstances, be much higher than those of other markers of oxidation such as lipid hydroperoxides....

Oxidatively modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Alzheimer's disease: many pathways to neurodegeneration.

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Butterfield, D Allan; Hardas, Sarita S; Lange, Miranda L Bader

2010-01-01

Recently, the oxidoreductase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has become a subject of interest as more and more studies reveal a surfeit of diverse GAPDH functions, extending beyond traditional aerobic metabolism of glucose. As a result of multiple isoforms and cellular locales, GAPDH is able to come in contact with a variety of small molecules, proteins, membranes, etc., that play important roles in normal and pathologic cell function. Specifically, GAPDH has been shown to interact with neurodegenerative disease-associated proteins, including the amyloid-beta protein precursor (AbetaPP). Studies from our laboratory have shown significant inhibition of GAPDH dehydrogenase activity in Alzheimer's disease (AD) brain due to oxidative modification. Although oxidative stress and damage is a common phenomenon in the AD brain, it would seem that inhibition of glycolytic enzyme activity is merely one avenue in which AD pathology affects neuronal cell development and survival, as oxidative modification can also impart a toxic gain-of-function to many proteins, including GAPDH. In this review, we examine the many functions of GAPDH with respect to AD brain; in particular, the apparent role(s) of GAPDH in AD-related apoptotic cell death is emphasized.

Impact of Post-Translational Modifications of Crop Proteins under Abiotic Stress

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Akiko Hashiguchi

2016-12-01

Full Text Available The efficiency of stress-induced adaptive responses of plants depends on intricate coordination of multiple signal transduction pathways that act coordinately or, in some cases, antagonistically. Protein post-translational modifications (PTMs can regulate protein activity and localization as well as protein–protein interactions in numerous cellular processes, thus leading to elaborate regulation of plant responses to various external stimuli. Understanding responses of crop plants under field conditions is crucial to design novel stress-tolerant cultivars that maintain robust homeostasis even under extreme conditions. In this review, proteomic studies of PTMs in crops are summarized. Although the research on the roles of crop PTMs in regulating stress response mechanisms is still in its early stage, several novel insights have been retrieved so far. This review covers techniques for detection of PTMs in plants, representative PTMs in plants under abiotic stress, and how PTMs control functions of representative proteins. In addition, because PTMs under abiotic stresses are well described in soybeans under submergence, recent findings in PTMs of soybean proteins under flooding stress are introduced. This review provides information on advances in PTM study in relation to plant adaptations to abiotic stresses, underlining the importance of PTM study to ensure adequate agricultural production in the future.

Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding.

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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.

PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications.

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Chaudhuri, Rima; Sadrieh, Arash; Hoffman, Nolan J; Parker, Benjamin L; Humphrey, Sean J; Stöckli, Jacqueline; Hill, Adam P; James, David E; Yang, Jean Yee Hwa

2015-08-19

Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog ( www.phosphortholog.com ) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community. Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites

Use of hydrostatic pressure for modulation of protein chemical modification and enzymatic selectivity.

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Makarov, Alexey A; Helmy, Roy; Joyce, Leo; Reibarkh, Mikhail; Maust, Mathew; Ren, Sumei; Mergelsberg, Ingrid; Welch, Christopher J

2016-05-11

Using hydrostatic pressure to induce protein conformational changes can be a powerful tool for altering the availability of protein reactive sites and for changing the selectivity of enzymatic reactions. Using a pressure apparatus, it has been demonstrated that hydrostatic pressure can be used to modulate the reactivity of lysine residues of the protein ubiquitin with a water-soluble amine-specific homobifunctional coupling agent. Fewer reactive lysine residues were observed when the reaction was carried out under elevated pressure of 3 kbar, consistent with a pressure-induced conformational change of ubiquitin that results in fewer exposed lysine residues. Additionally, modulation of the stereoselectivity of an enzymatic transamination reaction was observed at elevated hydrostatic pressure. In one case, the minor diasteromeric product formed at atmospheric pressure became the major product at elevated pressure. Such pressure-induced alterations of protein reactivity may provide an important new tool for enzymatic reactions and the chemical modification of proteins.

Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay.

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Weber, Alfred; Engelmaier, Andrea; Mohr, Gabriele; Haindl, Sonja; Schwarz, Hans Peter; Turecek, Peter L

2017-01-05

BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

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Hegele, Joerg [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)], E-mail: joerg.hegele@rdls.nestle.com; Buetler, Timo; Delatour, Thierry [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)

2008-06-09

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N{sup {epsilon}}-fructoselysine (FL), N{sup {epsilon}}-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 {+-} 3.81 nmol CML per {mu}mol of free Lys (Lys{sub free}) and 81.5 {+-} 87.8 nmol Pyr {mu}mol{sup -1} Lys{sub free}{sup -1} vs. 3.72 {+-} 1.29 nmol FL {mu}mol{sup -1} Lys{sub free}{sup -1}. In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 {+-} 0.08 nmol FL {mu}mol{sup -1} of protein-bound Lys (Lys{sub p-b}), 0.04 {+-} 0.03 nmol CML {mu}mol{sup -1} Lys{sub p-b}{sup -1} and 0.06 {+-} 0.02 nmol Pyr {mu}mol{sup -1} Lys{sub p-b}{sup -1}. It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

MORPHOLOGICAL RESEARCH ON FREE-RESIDUE OXIDATION PROCESSES IN CASES OF DECIDUAL CELLS OF PLACENTA IN CHORIOAMNIONOTIS AND BASAL DECIDUITIS COMBINED WITH IRON-DEFICIENCY ANEMIA IN THE PREGNANT

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V. V. Ilika

2017-07-01

Full Text Available Background. The oxidative modification of proteins is lately pivotal to pathologists and it is a new way of research on different pathological conditions, as well as the diagnostics of inflammation processes in placenta. Objective. The study was aimed at the research of nitro peroxides and establishing the specific features of oxidative modification of proteins in inflammation of placenta with iron deficient anaemia in the pregnant. Methods. Сhemiluminescent and histochemical technique (with bromphenol blue on ‘acidic’ and ‘basic’ proteins according to Mikel Calvo was applied. Results. The intensity of nitro peroxides glow in chorioamnionitis and basal deciduitis increased in comparison with the samples of physiological and iron deficient anaemia gestation. At the same time in chorioamnionitis the glow intensity is higher than in basal deciduitis. Due to the results of immune histochemical technique held while analysing the samples, together with chorioamnionitis and basal deciduitis the R/B increases and in basal deciduitis the rate, is probably, higher, than in chorioamnionitis. At the same time, the extent of oxidative modification of proteins in cases of inflammation with iron deficient anaemia in the pregnant is on the average higher than with no iron deficient anaemia in the pregnant. Conclusions. High level of nitro peroxides in placentae basal plate in secundines inflammation, the increase in R/B rate, in other words the prevalence of ‘acidic’ proteins over ‘basic’ ones, is evidenced due to the increase of the intensity of oxidative modification processes of proteins in cases of deciduitis.

Activation of cAMP-dependent signaling induces oxidative modification of the cardiac Na+-K+ pump and inhibits its activity.

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White, Caroline N; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Chia, Karin K M; Figtree, Gemma A; Rasmussen, Helge H

2010-04-30

Cellular signaling can inhibit the membrane Na(+)-K(+) pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the beta(1) subunit of the pump alpha/beta heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47(phox) and p22(phox), required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na(+)-K(+) pump beta(1) subunit and decreased its co-immunoprecipitation with the alpha(1) subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the alpha(1)/beta(1) subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the epsilon-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na(+)-K(+) pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump beta(1) subunit.

High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice.

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Kahle, M; Schäfer, A; Seelig, A; Schultheiß, J; Wu, M; Aichler, M; Leonhardt, J; Rathkolb, B; Rozman, J; Sarioglu, H; Hauck, S M; Ueffing, M; Wolf, E; Kastenmueller, G; Adamski, J; Walch, A; Hrabé de Angelis, M; Neschen, S

2015-01-01

Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and

An evaluation of heat on protein oxidation of soy protein isolate or soy protein isolate mixed with soybean oil and its consequences on redox status of broilers at early age

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Xianglun Zhang

2017-08-01

Full Text Available Objective The objective of this study was to evaluate effects of heat treatment and soybean oil inclusion on protein oxidation of soy protein isolate (SPI and of oxidized protein on redox status of broilers at an early age. Methods SPI mixed with soybean oil (SPIO heated at 100°C for 8 h was used to evaluate protein oxidation of SPI. A total of two hundred and sixteen 1-day-old Arbor Acres chicks were divided into 3 groups with 6 replicates of 12 birds, receiving basal diet (CON, heat-oxidized SPI diet (HSPI or mixture of SPI and 2% soybean oil diet (HSPIO for 21 d, respectively. Results Increased protein carbonyl, decreased protein sulfhydryl of SPI were observed as heating time increased in all treatments (p<0.05. Addition of 2% soybean oil increased protein carbonyl of SPI at 8 h heating (p<0.05. Dietary HSPI and HSPIO decreased the average daily gain of broilers as compared with the CON (p<0.05. Broilers fed HSPI and HSPIO exhibited decreased glutathione (GSH in serum, catalase activity and total sulfhydryl in liver and increased malondialdehyde (MDA and protein carbonyl in serum, advanced oxidation protein products (AOPPs in liver and protein carbonyl in jejunal mucosa as compared with that of the CON (p<0.05. Additionally, broilers receiving HSPIO showed decreased glutathione peroxidase activity (GSH-Px in serum, GSH and hydroxyl radical scavenging capacity in liver, GSH-Px activity in duodenal mucosa, GSH-Px activity and superoxide anion radical scavenging capacity in jejunal mucosa and increased AOPPs in serum, MDA and protein carbonyl in liver, MDA and AOPPs in jejunal mucosa (p<0.05. Conclusion Protein oxidation of SPI can be induced by heat and soybean oil and oxidized protein resulted in redox imbalance in broilers at an early age.

Engineering specific chemical modification sites into a collagen-like protein from Streptococcus pyogenes.

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Stoichevska, Violet; Peng, Yong Y; Vashi, Aditya V; Werkmeister, Jerome A; Dumsday, Geoff J; Ramshaw, John A M

2017-03-01

Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017. © 2016 Wiley Periodicals, Inc.

Oxidative proteome alterations during skeletal muscle ageing

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Sofia Lourenço dos Santos

2015-08-01

Full Text Available Sarcopenia corresponds to the degenerative loss of skeletal muscle mass, quality, and strength associated with ageing and leads to a progressive impairment of mobility and quality of life. However, the cellular and molecular mechanisms involved in this process are not completely understood. A hallmark of cellular and tissular ageing is the accumulation of oxidatively modified (carbonylated proteins, leading to a decreased quality of the cellular proteome that could directly impact on normal cellular functions. Although increased oxidative stress has been reported during skeletal muscle ageing, the oxidized protein targets, also referred as to the ‘oxi-proteome’ or ‘carbonylome’, have not been characterized yet. To better understand the mechanisms by which these damaged proteins build up and potentially affect muscle function, proteins targeted by these modifications have been identified in human rectus abdominis muscle obtained from young and old healthy donors using a bi-dimensional gel electrophoresis-based proteomic approach coupled with immunodetection of carbonylated proteins. Among evidenced protein spots, 17 were found as increased carbonylated in biopsies from old donors comparing to young counterparts. These proteins are involved in key cellular functions such as cellular morphology and transport, muscle contraction and energy metabolism. Importantly, impairment of these pathways has been described in skeletal muscle during ageing. Functional decline of these proteins due to irreversible oxidation may therefore impact directly on the above-mentioned pathways, hence contributing to the generation of the sarcopenic phenotype.

A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2

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Carlos R. Arias-Barreiro

2010-06-01

Full Text Available A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2. Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd2+, Cu2+, Pb2+, Zn2+ and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm were determined as 1.0 x 10−7 (arsenite, 1.0 x 10−4 (naphthalene, 1.0 x 10−4 (Cu2+, 3.8 x 10−4 (H2O2, 1.0 x 10−3 (Cd2+, 1.0 x 10−3 (Zn2+, 1.0 x 10−2 (menadione, 1.0 (triphenyltin, 1.56 (zinc pyrithione, 3.1 (selenite and 6.3 (Pb2+, respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation.

Oxidative modification of lipoic acid by HNE in Alzheimer disease brain

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Sarita S. Hardas

2013-01-01

Full Text Available Alzheimer disease (AD is an age-related neurodegenerative disease characterized by the presence of three pathological hallmarks: synapse loss, extracellular senile plaques (SP and intracellular neurofibrillary tangles (NFTs. The major component of SP is amyloid β-peptide (Aβ, which has been shown to induce oxidative stress. The AD brain shows increased levels of lipid peroxidation products, including 4-hydroxy-2-nonenal (HNE. HNE can react covalently with Cys, His, or Lys residues on proteins, altering structure and function of the latter. In the present study we measured the levels of the HNE-modified lipoic acid in brain of subjects with AD and age-matched controls. Lipoic acid is a key co-factor for a number of proteins including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, key complexes for cellular energetics. We observed a significant decrease in the levels of HNE-lipoic acid in the AD brain compared to that of age-matched controls. To investigate this phenomenon further, the levels and activity of lipoamide dehydrogenase (LADH were measured in AD and control brains. Additionally, LADH activities were measured after in-vitro HNE-treatment to mice brains. Both LADH levels and activities were found to be significantly reduced in AD brain compared to age-matched control. HNE-treatment also reduced the LADH activity in mice brain. These data are consistent with a two-hit hypothesis of AD: oxidative stress leads to lipid peroxidation that, in turn, causes oxidative dysfunction of key energy-related complexes in mitochondria, triggering neurodegeneration. This study is consonant with the notion that lipoic acid supplementation could be a potential treatment for the observed loss of cellular energetics in AD and potentiate the antioxidant defense system to prevent or delay the oxidative stress in and progression of this devastating dementing disorder.

The recombination protein RAD52 cooperates with the excision repair protein OGG1 for the repair of oxidative lesions in mammalian cells

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de Souza-Pinto, Nadja C; Maynard, Scott; Hashiguchi, Kazunari

2009-01-01

number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic...... knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1...... to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities....

The measurement of reversible redox dependent post-translational modifications and their regulation of mitochondrial and skeletal muscle function

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Philip A Kramer

2015-11-01

Full Text Available 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 how 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.

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

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

2015-11-25

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 how 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.

Oxidative Stress and Huntington's Disease: The Good, The Bad, and The Ugly.

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Kumar, Amit; Ratan, Rajiv R

2016-10-01

Redox homeostasis is crucial for proper cellular functions, including receptor tyrosine kinase signaling, protein folding, and xenobiotic detoxification. Under basal conditions, there is a balance between oxidants and antioxidants. This balance facilitates the ability of oxidants, such as reactive oxygen species, to play critical regulatory functions through a direct modification of a small number of amino acids (e.g. cysteine) on signaling proteins. These signaling functions leverage tight spatial, amplitude, and temporal control of oxidant concentrations. However, when oxidants overwhelm the antioxidant capacity, they lead to a harmful condition of oxidative stress. Oxidative stress has long been held to be one of the key players in disease progression for Huntington's disease (HD). In this review, we will critically review this evidence, drawing some intermediate conclusions, and ultimately provide a framework for thinking about the role of oxidative stress in the pathophysiology of HD.

Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques

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Woods, Alan A; Linton, Stuart M; Davies, Michael Jonathan

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...... 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...

Characterization of the Bat proteins in the oxidative stress response of Leptospira biflexa.

Science.gov (United States)

Stewart, Philip E; Carroll, James A; Dorward, David W; Stone, Hunter H; Sarkar, Amit; Picardeau, Mathieu; Rosa, Patricia A

2012-12-13

Leptospires lack many of the homologs for oxidative defense present in other bacteria, but do encode homologs of the Bacteriodes aerotolerance (Bat) proteins, which have been proposed to fulfill this function. Bat homologs have been identified in all families of the phylum Spirochaetes, yet a specific function for these proteins has not been experimentally demonstrated. We investigated the contribution of the Bat proteins in the model organism Leptospira biflexa for their potential contributions to growth rate, morphology and protection against oxidative challenges. A genetically engineered mutant strain in which all bat ORFs were deleted did not exhibit altered growth rate or morphology, relative to the wild-type strain. Nor could we demonstrate a protective role for the Bat proteins in coping with various oxidative stresses. Further, pre-exposing L. biflexa to sublethal levels of reactive oxygen species did not appear to induce a general oxidative stress response, in contrast to what has been shown in other bacterial species. Differential proteomic analysis of the wild-type and mutant strains detected changes in the abundance of a single protein only - HtpG, which is encoded by the gene immediately downstream of the bat loci. The data presented here do not support a protective role for the Leptospira Bat proteins in directly coping with oxidative stress as previously proposed. L. biflexa is relatively sensitive to reactive oxygen species such as superoxide and H2O2, suggesting that this spirochete lacks a strong, protective defense against oxidative damage despite being a strict aerobe.

Oxidative stress signaling to chromatin in health and disease

KAUST Repository

Kreuz, Sarah

2016-06-20

Oxidative stress has a significant impact on the development and progression of common human pathologies, including cancer, diabetes, hypertension and neurodegenerative diseases. Increasing evidence suggests that oxidative stress globally influences chromatin structure, DNA methylation, enzymatic and non-enzymatic post-translational modifications of histones and DNA-binding proteins. The effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including modulation of gene expression, cell death, cell survival and mutagenesis, which are disease-driving mechanisms in human pathologies. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment of these diseases. We summarize recent research developments connecting oxidative stress and chromatin regulation.

Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

Science.gov (United States)

Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

2016-04-01

We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Preparation and Biocompatible Surface Modification of Redox Altered Cerium Oxide Nanoparticle Promising for Nanobiology and Medicine

KAUST Repository

Nanda, Himansu Sekhar

2016-11-03

The biocompatible surface modification of metal oxide nanoparticles via surface functionalization technique has been used as an important tool in nanotechnology and medicine. In this report, we have prepared aqueous dispersible, trivalent metal ion (samarium)-doped cerium oxide nanoparticles (SmCNPs) as model redox altered CNPs of biological relevance. SmCNP surface modified with hydrophilic biocompatible (6-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-hexyl) triethoxysilane (MEEETES) were prepared using ammonia-induced ethylene glycol-assisted precipitation method and were characterized using a variety of complementary characterization techniques. The chemical interaction of functional moieties with the surface of doped nanoparticle was studied using powerful 13C cross polarization magic angle sample spinning nuclear magnetic resonance spectroscopy. The results demonstrated the production of the extremely small size MEEETES surface modified doped nanoparticles with significant reduction in aggregation compared to their unmodified state. Moreover, the functional moieties had strong chemical interaction with the surface of the doped nanoparticles. The biocompatible surface modification using MEEETES should also be extended to several other transition metal ion doped and co-doped CNPs for the production of aqueous dispersible redox altered CNPs that are promising for nanobiology and medicine.

Preparation and Biocompatible Surface Modification of Redox Altered Cerium Oxide Nanoparticle Promising for Nanobiology and Medicine

KAUST Repository

Nanda, Himansu Sekhar

2016-01-01

The biocompatible surface modification of metal oxide nanoparticles via surface functionalization technique has been used as an important tool in nanotechnology and medicine. In this report, we have prepared aqueous dispersible, trivalent metal ion (samarium)-doped cerium oxide nanoparticles (SmCNPs) as model redox altered CNPs of biological relevance. SmCNP surface modified with hydrophilic biocompatible (6-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-hexyl) triethoxysilane (MEEETES) were prepared using ammonia-induced ethylene glycol-assisted precipitation method and were characterized using a variety of complementary characterization techniques. The chemical interaction of functional moieties with the surface of doped nanoparticle was studied using powerful 13C cross polarization magic angle sample spinning nuclear magnetic resonance spectroscopy. The results demonstrated the production of the extremely small size MEEETES surface modified doped nanoparticles with significant reduction in aggregation compared to their unmodified state. Moreover, the functional moieties had strong chemical interaction with the surface of the doped nanoparticles. The biocompatible surface modification using MEEETES should also be extended to several other transition metal ion doped and co-doped CNPs for the production of aqueous dispersible redox altered CNPs that are promising for nanobiology and medicine.

Preparation and Biocompatible Surface Modification of Redox Altered Cerium Oxide Nanoparticle Promising for Nanobiology and Medicine

Directory of Open Access Journals (Sweden)

Himansu Sekhar Nanda

2016-11-01

Full Text Available The biocompatible surface modification of metal oxide nanoparticles via surface functionalization technique has been used as an important tool in nanotechnology and medicine. In this report, we have prepared aqueous dispersible, trivalent metal ion (samarium-doped cerium oxide nanoparticles (SmCNPs as model redox altered CNPs of biological relevance. SmCNP surface modified with hydrophilic biocompatible (6-{2-[2-(2-methoxy-ethoxy-ethoxy]-ethoxy}-hexyl triethoxysilane (MEEETES were prepared using ammonia-induced ethylene glycol-assisted precipitation method and were characterized using a variety of complementary characterization techniques. The chemical interaction of functional moieties with the surface of doped nanoparticle was studied using powerful 13C cross polarization magic angle sample spinning nuclear magnetic resonance spectroscopy. The results demonstrated the production of the extremely small size MEEETES surface modified doped nanoparticles with significant reduction in aggregation compared to their unmodified state. Moreover, the functional moieties had strong chemical interaction with the surface of the doped nanoparticles. The biocompatible surface modification using MEEETES should also be extended to several other transition metal ion doped and co-doped CNPs for the production of aqueous dispersible redox altered CNPs that are promising for nanobiology and medicine.

Oxidative stress and CCN1 protein in human skin connective tissue aging

Directory of Open Access Journals (Sweden)

Zhaoping Qin

2016-06-01

Full Text Available Reactive oxygen species (ROS is an important pathogenic factor involved in human aging. Human skin is a primary target of oxidative stress from ROS generated from both extrinsic and intrinsic sources, like ultraviolet irradiation (UV and endogenous oxidative metabolism. Oxidative stress causes the alterations of collagen-rich extracellular matrix (ECM, the hallmark of skin connective tissue aging. Age-related alteration of dermal collagenous ECM impairs skin structural integrity and creates a tissue microenvironment that promotes age-related skin diseases, such as poor wound healing and skin cancer. Here, we review recent advances in our understanding of oxidative stress and CCN1 protein (first member of CCN family proteins, a critical mediator of oxidative stress-induced skin connective tissue aging.

Glycan Reader is improved to recognize most sugar types and chemical modifications in the Protein Data Bank.

Science.gov (United States)

Park, Sang-Jun; Lee, Jumin; Patel, Dhilon S; Ma, Hongjing; Lee, Hui Sun; Jo, Sunhwan; Im, Wonpil

2017-10-01

Glycans play a central role in many essential biological processes. Glycan Reader was originally developed to simplify the reading of Protein Data Bank (PDB) files containing glycans through the automatic detection and annotation of sugars and glycosidic linkages between sugar units and to proteins, all based on atomic coordinates and connectivity information. Carbohydrates can have various chemical modifications at different positions, making their chemical space much diverse. Unfortunately, current PDB files do not provide exact annotations for most carbohydrate derivatives and more than 50% of PDB glycan chains have at least one carbohydrate derivative that could not be correctly recognized by the original Glycan Reader. Glycan Reader has been improved and now identifies most sugar types and chemical modifications (including various glycolipids) in the PDB, and both PDB and PDBx/mmCIF formats are supported. CHARMM-GUI Glycan Reader is updated to generate the simulation system and input of various glycoconjugates with most sugar types and chemical modifications. It also offers a new functionality to edit the glycan structures through addition/deletion/modification of glycosylation types, sugar types, chemical modifications, glycosidic linkages, and anomeric states. The simulation system and input files can be used for CHARMM, NAMD, GROMACS, AMBER, GENESIS, LAMMPS, Desmond, OpenMM, and CHARMM/OpenMM. Glycan Fragment Database in GlycanStructure.Org is also updated to provide an intuitive glycan sequence search tool for complex glycan structures with various chemical modifications in the PDB. http://www.charmm-gui.org/input/glycan and http://www.glycanstructure.org. wonpil@lehigh.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

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Catarina Moreirinha

2018-03-01

Full Text Available The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells.

Time dynamics of the Bacillus cereus exoproteome are shaped by cellular oxidation

Directory of Open Access Journals (Sweden)

Jean-Paul eMadeira

2015-04-01

Full Text Available At low density, Bacillus cereus cells release a large variety of proteins into the extracellular medium when cultivated in pH-regulated, glucose-containing minimal medium, either in the presence or absence of oxygen. The majority of these exoproteins are putative virulence factors, including toxin-related proteins. Here, B. cereus exoproteome time courses were monitored by nanoLC-MS/MS under low-oxidoreduction potential (ORP anaerobiosis, high-ORP anaerobiosis, and aerobiosis, with a specific focus on oxidative-induced post-translational modifications of methionine residues. Principal component analysis (PCA of the exoproteome dynamics indicated that toxin-related proteins were the most representative of the exoproteome changes, both in terms of protein abundance and their methionine sulfoxide (Met(O content. PCA also revealed an interesting interconnection between toxin-, metabolism-, and oxidative stress–related proteins, suggesting that the abundance level of toxin-related proteins, and their Met(O content in the B. cereus exoproteome, reflected the cellular oxidation under both aerobiosis and anaerobiosis.

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

Science.gov (United States)

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2016-09-01

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. Copyright © 2016 Elsevier Ltd. All rights reserved.

O-GlcNAc modification of the coat protein of the potyvirus Plum pox virus enhances viral infection.

Science.gov (United States)

Pérez, José de Jesús; Udeshi, Namrata D; Shabanowitz, Jeffrey; Ciordia, Sergio; Juárez, Silvia; Scott, Cheryl L; Olszewski, Neil E; Hunt, Donald F; García, Juan Antonio

2013-08-01

O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus. Copyright © 2013 Elsevier Inc. All rights reserved.

DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

Science.gov (United States)

Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

2013-01-01

Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

β(3) adrenergic stimulation of the cardiac Na+-K+ pump by reversal of an inhibitory oxidative modification.

Science.gov (United States)

Bundgaard, Henning; Liu, Chia-Chi; Garcia, Alvaro; Hamilton, Elisha J; Huang, Yifei; Chia, Karin K M; Hunyor, Stephen N; Figtree, Gemma A; Rasmussen, Helge H

2010-12-21

inhibition of L-type Ca(2+) current contributes to negative inotropy of β(3) adrenergic receptor (β(3) AR) activation, but effects on other determinants of excitation-contraction coupling are not known. Of these, the Na(+)-K(+) pump is of particular interest because of adverse effects attributed to high cardiac myocyte Na(+) levels and upregulation of the β(3) AR in heart failure. we voltage clamped rabbit ventricular myocytes and identified electrogenic Na(+)-K(+) pump current (I(p)) as the shift in holding current induced by ouabain. The synthetic β(3) AR agonists BRL37344 and CL316,243 and the natural agonist norepinephrine increased I(p). Pump stimulation was insensitive to the β(1)/β(2) AR antagonist nadolol and the protein kinase A inhibitor H-89 but sensitive to the β(3) AR antagonist L-748,337. Blockade of nitric oxide synthase abolished pump stimulation and an increase in fluorescence of myocytes loaded with a nitric oxide-sensitive dye. Exposure of myocytes to β(3) AR agonists decreased β(1) Na(+)-K(+) pump subunit glutathionylation, an oxidative modification that causes pump inhibition. The in vivo relevance of this was indicated by an increase in myocardial β(1) pump subunit glutathionylation with elimination of β(3) AR-mediated signaling in β(3) AR(-/-) mice. The in vivo effect of BRL37344 on contractility of the nonfailing and failing heart in sheep was consistent with a beneficial effect of Na(+)-K(+) pump stimulation in heart failure. the β(3) AR mediates decreased β(1) subunit glutathionylation and Na(+)-K(+) pump stimulation in the heart. Upregulation of the receptor in heart failure may be a beneficial mechanism that facilitates the export of excess Na(+).

Oxidative Stress in the Carcinogenicity of Chemical Carcinogens

International Nuclear Information System (INIS)

Kakehashi, Anna; Wei, Min; Fukushima, Shoji; Wanibuchi, Hideki

2013-01-01

This review highlights several in vivo studies utilizing non-genotoxic and genotoxic chemical carcinogens, and the mechanisms of their high and low dose carcinogenicities with respect to formation of oxidative stress. Here, we survey the examples and discuss possible mechanisms of hormetic effects with cytochrome P 450 inducers, such as phenobarbital, α-benzene hexachloride and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane. Epigenetic processes differentially can be affected by agents that impinge on oxidative DNA damage, repair, apoptosis, cell proliferation, intracellular communication and cell signaling. Non-genotoxic carcinogens may target nuclear receptors and induce post-translational modifications at the protein level, thereby impacting on the stability or activity of key regulatory proteins, including oncoproteins and tumor suppressor proteins. We further discuss role of oxidative stress focusing on the low dose carcinogenicities of several genotoxic carcinogens such as a hepatocarcinogen contained in seared fish and meat, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, arsenic and its metabolites, and the kidney carcinogen potassium bromate

Oxidative Stress in the Carcinogenicity of Chemical Carcinogens

Energy Technology Data Exchange (ETDEWEB)

Kakehashi, Anna; Wei, Min [Department of Pathology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-Ku, Osaka 545-8585 (Japan); Fukushima, Shoji [Japan Bioassay Research Center, Japan Industrial Safety and Health Association, 2445 Hirasawa, Hadano, Kanagawa 257-0015 (Japan); Wanibuchi, Hideki, E-mail: wani@med.osaka-cu.ac.jp [Department of Pathology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-Ku, Osaka 545-8585 (Japan)

2013-10-28

This review highlights several in vivo studies utilizing non-genotoxic and genotoxic chemical carcinogens, and the mechanisms of their high and low dose carcinogenicities with respect to formation of oxidative stress. Here, we survey the examples and discuss possible mechanisms of hormetic effects with cytochrome P{sub 450} inducers, such as phenobarbital, α-benzene hexachloride and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane. Epigenetic processes differentially can be affected by agents that impinge on oxidative DNA damage, repair, apoptosis, cell proliferation, intracellular communication and cell signaling. Non-genotoxic carcinogens may target nuclear receptors and induce post-translational modifications at the protein level, thereby impacting on the stability or activity of key regulatory proteins, including oncoproteins and tumor suppressor proteins. We further discuss role of oxidative stress focusing on the low dose carcinogenicities of several genotoxic carcinogens such as a hepatocarcinogen contained in seared fish and meat, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, arsenic and its metabolites, and the kidney carcinogen potassium bromate.

A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

DEFF Research Database (Denmark)

Pirone, Lucia; Xolalpa, Wendy; Sigurdsson, Jón Otti

2017-01-01

L conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bio...

Quantitative analysis of histone modifications: formaldehyde is a source of pathological n(6-formyllysine that is refractory to histone deacetylases.

Directory of Open Access Journals (Sweden)

Bahar Edrissi

Full Text Available Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N(6-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3'-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N(6-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N(6-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N(6-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1-4 modifications per 10(4 lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10(4 lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N(6-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6-formyllysine, with use of [(13C,(2H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10% with a peptide substrate containing the formyl adduct. These data suggest that N(6-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary

Modification of the surface energy in isovalent nano-oxides prepared by chemical synthesis

International Nuclear Information System (INIS)

Miagava, J.; Gouvea, D.

2011-01-01

The phase stability of the nano-oxides depends on the bulk energy but it also depends on the surface energy. The difference of surface energy of the rutile and anatase phases result in a change of phase stability: TiO_2 without additives is stable as anatase when particles have nanometric size and a high specific surface area whereas rutile is stable when particles are larger. But this stability can be modified through the use of additives. Different studies demonstrate that additives segregate on the particle surface modifying the surface energy. In this work (1-X)TiO_2-XSnO_2 powders were synthesized by the polymeric precursor method with concentrations of 0 ≤ X ≤ 1. The specific surface area measurements demonstrate that the modification of the composition change the specific surface areas and it reaches a maximum at X = 0.005. The Raman spectroscopy demonstrates that a modification on the stability of the TiO_2 polymorphs occurs and the phase rutile is stabilized when SnO_2 is added to the nano powders.(author)

Seasonal variability of oxidative stress markers in city bus drivers. Part II. Oxidative damage to lipids and proteins.

Science.gov (United States)

Rossner, Pavel; Svecova, Vlasta; Milcova, Alena; Lnenickova, Zdena; Solansky, Ivo; Sram, Radim J

2008-07-03

The aim of the present study was to investigate the seasonal variability of markers of oxidative damage to lipids (15-F2t-isoprostane, 15-F2t-IsoP) and proteins (protein carbonyl levels) in 50 bus drivers and 50 controls from Prague, Czech Republic, and to identify factors affecting oxidative stress markers. The samples were collected in three seasons with different levels of air pollution. The exposure to environmental pollutants (carcinogenic polycyclic aromatic hydrocarbons, c-PAHs, particulate matter, PM2.5 and PM10, and volatile organic compounds, VOC) was monitored by personal and/or stationary monitors. For the analysis of both markers, ELISA techniques were used. The median levels of individual markers in bus drivers versus controls were as follows: 15-F2t-IsoP (nmol/mmol creatinine): winter 2005, 0.81 versus 0.68 (pbus drivers in winter seasons, but not in summer. Lipid peroxidation was positively correlated with c-PAHs and PM exposure; protein oxidation correlated negatively and was highest in summer suggesting another factor(s) affecting protein carbonyl levels.

Recent advances in synthesis and surface modification of superparamagnetic iron oxide nanoparticles with silica

Energy Technology Data Exchange (ETDEWEB)

Sodipo, Bashiru Kayode, E-mail: bashirsodipo@gmail.com [School of Physics, Universiti Sains Malaysia, 11800 Pulau Pinang (Malaysia); Nano-Biotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Pulau Pinang (Malaysia); Aziz, Azlan Abdul [School of Physics, Universiti Sains Malaysia, 11800 Pulau Pinang (Malaysia); Nano-Biotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Pulau Pinang (Malaysia)

2016-10-15

Research on synthesis of superparamagnetic iron oxide nanoparticles (SPION) and its surface modification for biomedical applications is of intense interest. Due to superparamagnetic property of SPION, the nanoparticles have large magnetic susceptibility, single magnetic domain and controllable magnetic behaviour. However, owing to easy agglomeration of SPION, surface modification of the magnetic particles with biocompatible materials such as silica nanoparticle has gained much attention in the last decade. In this review, we present recent advances in synthesis of SPION and various routes of producing silica coated SPION. - Highlights: • We present recent advances in synthesis of SPION and various routes of producing silica coated SPION • The synthetic routes of producing SPION can be classified into three: physical, chemical and biological methods. • The chemical method is the most cited method of producing SPION and it sub-classified into liquid and gas phase. • The techniques of producing silica coated SPION is grouped into seeded and non-seeded methods.

Recent advances in synthesis and surface modification of superparamagnetic iron oxide nanoparticles with silica

International Nuclear Information System (INIS)

Sodipo, Bashiru Kayode; Aziz, Azlan Abdul

2016-01-01

Research on synthesis of superparamagnetic iron oxide nanoparticles (SPION) and its surface modification for biomedical applications is of intense interest. Due to superparamagnetic property of SPION, the nanoparticles have large magnetic susceptibility, single magnetic domain and controllable magnetic behaviour. However, owing to easy agglomeration of SPION, surface modification of the magnetic particles with biocompatible materials such as silica nanoparticle has gained much attention in the last decade. In this review, we present recent advances in synthesis of SPION and various routes of producing silica coated SPION. - Highlights: • We present recent advances in synthesis of SPION and various routes of producing silica coated SPION • The synthetic routes of producing SPION can be classified into three: physical, chemical and biological methods. • The chemical method is the most cited method of producing SPION and it sub-classified into liquid and gas phase. • The techniques of producing silica coated SPION is grouped into seeded and non-seeded methods.

O-GlcNAc modification: why so intimately associated with phosphorylation?

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Ande Sudharsana R

2011-01-01

Full Text Available Abstract Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68% than its normal occurrence (~2% alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

The SNO/SOH TMT strategy for combinatorial analysis of reversible cysteine oxidations

DEFF Research Database (Denmark)

Wojdyla, Katarzyna; Williamson, James; Roepstorff, Peter

2015-01-01

UNLABELLED: Redox homeostasis is essential for normal function of cells and redox imbalance has been recognised as a pathogenic factor of numerous human diseases. Oxidative modifications of cysteine thiols modulate function of many proteins, mediate signalling, and fine-tune transcriptional...... on commercially available reagents. The method has proven precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale....

Nanoscale surface modification of Li-rich layered oxides for high-capacity cathodes in Li-ion batteries

Science.gov (United States)

Lan, Xiwei; Xin, Yue; Wang, Libin; Hu, Xianluo

2018-03-01

Li-rich layered oxides (LLOs) have been developed as a high-capacity cathode material for Li-ion batteries, but the structural complexity and unique initial charging behavior lead to several problems including large initial capacity loss, capacity and voltage fading, poor cyclability, and inferior rate capability. Since the surface conditions are critical to electrochemical performance and the drawbacks, nanoscale surface modification for improving LLO's properties is a general strategy. This review mainly summarizes the surface modification of LLOs and classifies them into three types of surface pre-treatment, surface gradient doping, and surface coating. Surface pre-treatment usually introduces removal of Li2O for lower irreversible capacity while surface doping is aimed to stabilize the structure during electrochemical cycling. Surface coating layers with different properties, protective layers to suppress the interface side reaction, coating layers related to structural transformation, and electronic/ionic conductive layers for better rate capability, can avoid the shortcomings of LLOs. In addition to surface modification for performance enhancement, other strategies can also be investigated to achieve high-performance LLO-based cathode materials.

Cross-linking by protein oxidation in the rapidly setting gel-based glues of slugs

Science.gov (United States)

Bradshaw, Andrew; Salt, Michael; Bell, Ashley; Zeitler, Matt; Litra, Noelle; Smith, Andrew M.

2011-01-01

SUMMARY The terrestrial slug Arion subfuscus secretes a glue that is a dilute gel with remarkable adhesive and cohesive strength. The function of this glue depends on metals, raising the possibility that metal-catalyzed oxidation plays a role. The extent and time course of protein oxidation was measured by immunoblotting to detect the resulting carbonyl groups. Several proteins, particularly one with a relative molecular mass (Mr) of 165×103, were heavily oxidized. Of the proteins known to distinguish the glue from non-adhesive mucus, only specific size variants were oxidized. The oxidation appears to occur within the first few seconds of secretion. Although carbonyls were detected by 2,4-dinitrophenylhydrazine (DNPH) in denatured proteins, they were not easily detected in the native state. The presence of reversible cross-links derived from carbonyls was tested for by treatment with sodium borohydride, which would reduce uncross-linked carbonyls to alcohols, but stabilize imine bonds formed by carbonyls and thus lead to less soluble complexes. Consistent with imine bond formation, sodium borohydride led to a 20–35% decrease in the amount of soluble protein with a Mr of 40–165 (×103) without changing the carbonyl content per protein. In contrast, the nucleophile hydroxylamine, which would competitively disrupt imine bonds, increased protein solubility in the glue. Finally, the primary amine groups on a protein with a Mr of 15×103 were not accessible to acid anhydrides. The results suggest that cross-links between aldehydes and primary amines contribute to the cohesive strength of the glue. PMID:21525316

Design of Highly Selective Gas Sensors via Physicochemical Modification of Oxide Nanowires: Overview

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Hyung-Sik Woo

2016-09-01

Full Text Available Strategies for the enhancement of gas sensing properties, and specifically the improvement of gas selectivity of metal oxide semiconductor nanowire (NW networks grown by chemical vapor deposition and thermal evaporation, are reviewed. Highly crystalline NWs grown by vapor-phase routes have various advantages, and thus have been applied in the field of gas sensors over the years. In particular, n-type NWs such as SnO2, ZnO, and In2O3 are widely studied because of their simple synthetic preparation and high gas response. However, due to their usually high responses to C2H5OH and NO2, the selective detection of other harmful and toxic gases using oxide NWs remains a challenging issue. Various strategies—such as doping/loading of noble metals, decorating/doping of catalytic metal oxides, and the formation of core–shell structures—have been explored to enhance gas selectivity and sensitivity, and are discussed herein. Additional methods such as the transformation of n-type into p-type NWs and the formation of catalyst-doped hierarchical structures by branch growth have also proven to be promising for the enhancement of gas selectivity. Accordingly, the physicochemical modification of oxide NWs via various methods provides new strategies to achieve the selective detection of a specific gas, and after further investigations, this approach could pave a new way in the field of NW-based semiconductor-type gas sensors.

Oxidative changes in lipids, proteins, and antioxidants in yogurt during the shelf life.

Science.gov (United States)

Citta, Anna; Folda, Alessandra; Scalcon, Valeria; Scutari, Guido; Bindoli, Alberto; Bellamio, Marco; Feller, Emiliano; Rigobello, Maria Pia

2017-11-01

Oxidation processes in milk and yogurt during the shelf life can result in an alteration of protein and lipid constituents. Therefore, the antioxidant properties of yogurt in standard conditions of preservation were evaluated. Total phenols, free radical scavenger activity, degree of lipid peroxidation, and protein oxidation were determined in plain and skim yogurts with or without fruit puree. After production, plain, skim, plain berries, and skim berries yogurts were compared during the shelf life up to 9 weeks. All types of yogurts revealed a basal antioxidant activity that was higher when a fruit puree was present but gradually decreased during the shelf life. However, after 5-8 weeks, antioxidant activity increased again. Both in plain and berries yogurts lipid peroxidation increased until the seventh week of shelf life and after decreased, whereas protein oxidation of all yogurts was similar either in the absence or presence of berries and increased during shelf life. During the shelf life, a different behavior between lipid and protein oxidation takes place and the presence of berries determines a protection only against lipid peroxidation.

Protein Oxidation in the Lungs of C57BL/6J Mice Following X-Irradiation

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Barshishat-Kupper, Michal; McCart, Elizabeth A.; Freedy, James G.; Tipton, Ashlee J.; Nagy, Vitaly; Kim, Sung-Yop; Landauer, Michael R.; Mueller, Gregory P.; Day, Regina M.

2015-01-01

Damage to normal lung tissue is a limiting factor when ionizing radiation is used in clinical applications. In addition, radiation pneumonitis and fibrosis are a major cause of mortality following accidental radiation exposure in humans. Although clinical symptoms may not develop for months after radiation exposure, immediate events induced by radiation are believed to generate molecular and cellular cascades that proceed during a clinical latent period. Oxidative damage to DNA is considered a primary cause of radiation injury to cells. DNA can be repaired by highly efficient mechanisms while repair of oxidized proteins is limited. Oxidized proteins are often destined for degradation. We examined protein oxidation following 17 Gy (0.6 Gy/min) thoracic X-irradiation in C57BL/6J mice. Seventeen Gy thoracic irradiation resulted in 100% mortality of mice within 127–189 days postirradiation. Necropsy findings indicated that pneumonitis and pulmonary fibrosis were the leading cause of mortality. We investigated the oxidation of lung proteins at 24 h postirradiation following 17 Gy thoracic irradiation using 2-D gel electrophoresis and OxyBlot for the detection of protein carbonylation. Seven carbonylated proteins were identified using mass spectrometry: serum albumin, selenium binding protein-1, alpha antitrypsin, cytoplasmic actin-1, carbonic anhydrase-2, peroxiredoxin-6, and apolipoprotein A1. The carbonylation status of carbonic anhydrase-2, selenium binding protein, and peroxiredoxin-6 was higher in control lung tissue. Apolipoprotein A1 and serum albumin carbonylation were increased following X-irradiation, as confirmed by OxyBlot immunoprecipitation and Western blotting. Our findings indicate that the profile of specific protein oxidation in the lung is altered following radiation exposure. PMID:28248270

Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

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Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

2017-05-01

We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

Graphene oxide as a protein matrix: influence on protein biophysical properties.

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Hernández-Cancel, Griselle; Suazo-Dávila, Dámaris; Ojeda-Cruzado, Axel J; García-Torres, Desiree; Cabrera, Carlos R; Griebenow, Kai

2015-10-19

This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.

[Effects of metal-catalyzed oxidation on the formation of advanced oxidation protein products].

Science.gov (United States)

Li, Li; Peng, Ai; Zhu, Kai-Yuan; Yu, Hong; Ll, Xin-Hua; Li, Chang-Bin

2008-03-11

To explore the relationship between metal-catalyzed oxidation (MCO) and the formation of advanced oxidation protein products (AOPPs). Specimens of human serum albumin (HSA) and pooled plasma were collected from 3 healthy volunteers and 4 uremia patients were divided into 3 groups: Group A incubated with copper sulfate solution of the concentrations of 0, 0.2, or 0.5 mmol/L, Group B, incubated with hydrogen peroxide 2 mmol/L, and Group C, incubated with copper sulfate 0.2 or 0.5 mmol/L plus hydrogen peroxide 2 mmol/L. 30 min and 24 h later the AOPP level was determined by ultraviolet visible spectrophotometry. High-performance liquid chromatography (HPLC) was used to observe the fragmentation effect on plasma proteins. Ninhydrin method was used to examine the protein fragments. The scavenging capacity of hydroxyl radical by macromolecules was measured so as to estimate the extent of damage for proteins induced by MCO. (1) The AOPP level of the HSA and plasma specimens of the uremia patients increased along with the increase of cupric ion concentration in a dose-dependent manner, especially in the presence of hydrogen peroxide (P < 0.05). (2) Aggregation of proteins was almost negligible in all groups, however, HPLC showed that cupric ion with or without hydrogen peroxide increased the fragments in the HAS specimens (with a relative molecular mass of 5000) and uremia patients' plasma proteins (with the molecular mass 7000). (3) The plasma AOPP level of the healthy volunteers was 68.2 micromol/L +/- 2.4 micromol/L, significantly lower than that of the uremia patients (158.5 micromol/L +/- 8.2 micromol/L). (4) The scavenging ability to clear hydroxyl radical by plasma proteins of the healthy volunteers was 1.38 -9.03 times as higher than that of the uremia patients. MCO contributes to the formation of AOPPs mainly through its fragmentation effect to proteins.

Oxidative Stress in Oral Diseases: Understanding Its Relation with Other Systemic Diseases

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Jaya Kumar

2017-09-01

Full Text Available Oxidative stress occurs in diabetes, various cancers, liver diseases, stroke, rheumatoid arthritis, chronic inflammation, and other degenerative diseases related to the nervous system. The free radicals have deleterious effect on various organs of the body. This is due to lipid peroxidation and irreversible protein modification that leads to cellular apoptosis or programmed cell death. During recent years, there is a rise in the oral diseases related to oxidative stress. Oxidative stress in oral disease is related to other systemic diseases in the body such as periodontitis, cardiovascular, pancreatic, gastric, and liver diseases. In the present review, we discuss the various pathways that mediate oxidative cellular damage. Numerous pathways mediate oxidative cellular damage and these include caspase pathway, PERK/NRF2 pathway, NADPH oxidase 4 pathways and JNK/mitogen-activated protein (MAP kinase pathway. We also discuss the role of inflammatory markers, lipid peroxidation, and role of oxygen species linked to oxidative stress. Knowledge of different pathways, role of inflammatory markers, and importance of low-density lipoprotein, fibrinogen, creatinine, nitric oxide, nitrates, and highly sensitive C-reactive proteins may be helpful in understanding the pathogenesis and plan better treatment for oral diseases which involve oxidative stress.

Peptidyl-prolyl cis/trans-isomerase A1 (Pin1) is a target for modification by lipid electrophiles.

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Aluise, Christopher D; Rose, Kristie; Boiani, Mariana; Reyzer, Michelle L; Manna, Joseph D; Tallman, Keri; Porter, Ned A; Marnett, Lawrence J

2013-02-18

Oxidation of membrane phospholipids is associated with inflammation, neurodegenerative disease, and cancer. Oxyradical damage to phospholipids results in the production of reactive aldehydes that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we demonstrate that peptidyl-prolyl cis/trans-isomerase A1 (Pin1), an enzyme that catalyzes the conversion of the peptide bond of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry resulted in detection of Michael adducts at the active site residues His-157 and Cys-113. Time and concentration dependencies indicate that Cys-113 is the primary site of HNE modification. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and Western blotting with anti-Pin1 antibody. Furthermore, orbitrap MS data support the adduction of Cys-113 in the Pin1 active site upon HNE treatment of MDA-MB-231 cells. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation.

Ammonia modification of activated carbon to enhance carbon dioxide adsorption: Effect of pre-oxidation

Science.gov (United States)

Shafeeyan, Mohammad Saleh; Daud, Wan Mohd Ashri Wan; Houshmand, Amirhossein; Arami-Niya, Arash

2011-02-01

A commercial granular activated carbon (GAC) was subjected to thermal treatment with ammonia for obtaining an efficient carbon dioxide (CO2) adsorbent. In general, CO2 adsorption capacity of activated carbon can be increased by introduction of basic nitrogen functionalities onto the carbon surface. In this work, the effect of oxygen surface groups before introduction of basic nitrogen functionalities to the carbon surface on CO2 adsorption capacity was investigated. For this purpose two different approaches of ammonia treatment without preliminary oxidation and amination of oxidized samples were studied. Modified carbons were characterized by elemental analysis and Fourier Transform Infrared spectroscopy (FT-IR) to study the impact of changes in surface chemistry and formation of specific surface groups on adsorption properties. The texture of the samples was characterized by conducting N2 adsorption/desorption at -196 °C. CO2 capture performance of the samples was investigated using a thermogravimetric analysis (TGA). It was found that in both modification techniques, the presence of nitrogen functionalities on carbon surface generally increased the CO2 adsorption capacity. The results indicated that oxidation followed by high temperature ammonia treatment (800 °C) considerably enhanced the CO2 uptake at higher temperatures.

Oxidation of lipid and protein in horse mackerel (Trachurus trachurus) mince and washed minces during processing and storage

DEFF Research Database (Denmark)

Eymard, Sylvie; Baron, Caroline; Jacobsen, Charlotte

2009-01-01

: M1, M2 and M3, with one, two and three washing steps, respectively. The different products were characterised (i.e. lipid content, protein, water, iron, fatty acid profile and tocopherol content) and analysed for protein and lipid oxidation in order to investigate the impact of the washing steps...... was followed by determination of protein solubility, protein thiol groups and protein carbonyl groups using colorimetric methods as well as western blotting for protein carbonyl groups. Lipid and protein oxidation markers indicated that both lipid and protein oxidation took place during processing...

Methionine sulfoxide profiling of milk proteins to assess the influence of lipids on protein oxidation in milk.

Science.gov (United States)

Wüst, Johannes; Pischetsrieder, Monika

2016-06-15

Thermal treatment of milk and milk products leads to protein oxidation, mainly the formation of methionine sulfoxide. Reactive oxygen species, responsible for the oxidation, can be generated by Maillard reaction, autoxidation of sugars, or lipid peroxidation. The present study investigated the influence of milk fat on methionine oxidation in milk. For this purpose, quantitative methionine sulfoxide profiling of all ten methionine residues of β-lactoglobulin, α-lactalbumin, and αs1-casein was carried out by ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry with scheduled multiple reaction monitoring (UHPLC-ESI-MS/MS-sMRM). Analysis of defatted and regular raw milk samples after heating for up to 8 min at 120 °C and analysis of ultrahigh-temperature milk samples with 0.1%, 1.5%, and 3.5% fat revealed that methionine oxidation of the five residues of the whey proteins and of residues M 123, M 135, and M 196 of αs1-casein was not affected or even suppressed in the presence of milk fat. Only the oxidation of residues M 54 and M 60 of αs1-casein was promoted by lipids. In evaporated milk samples, formation of methionine sulfoxide was hardly influenced by the fat content of the samples. Thus, it can be concluded that lipid oxidation products are not the major cause of methionine oxidation in milk.

Role of nitric oxide in the radiation-induced bystander effect.

Science.gov (United States)

Yakovlev, Vasily A

2015-12-01

Cells that are not irradiated but are affected by "stress signal factors" released from irradiated cells are called bystander cells. These cells, as well as directly irradiated ones, express DNA damage-related proteins and display excess DNA damage, chromosome aberrations, mutations, and malignant transformation. This phenomenon has been studied widely in the past 20 years, since its first description by Nagasawa and Little in 1992, and is known as the radiation-induced bystander effect (RIBE). Several factors have been identified as playing a role in the bystander response. This review will focus on one of them, nitric oxide (NO), and its role in the stimulation and propagation of RIBE. The hydrophobic properties of NO, which permit its diffusion through the cytoplasm and plasma membranes, allow this signaling molecule to easily spread from irradiated cells to bystander cells without the involvement of gap junction intercellular communication. NO produced in irradiated tissues mediates cellular regulation through posttranslational modification of a number of regulatory proteins. The best studied of these modifications are S-nitrosylation (reversible oxidation of cysteine) and tyrosine nitration. These modifications can up- or down-regulate the functions of many proteins modulating different NO-dependent effects. These NO-dependent effects include the stimulation of genomic instability (GI) and the accumulation of DNA errors in bystander cells without direct DNA damage. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Capsid protein oxidation in feline calicivirus using an electrochemical inactivation treatment

Energy Technology Data Exchange (ETDEWEB)

Shionoiri, Nozomi; Nogariya, Osamu; Tanaka, Masayoshi; Matsunaga, Tadashi; Tanaka, Tsuyoshi, E-mail: tsuyo@cc.tuat.ac.jp

2015-02-11

Highlights: • Feline calicivirus was inactivated electrochemically by a factor of >5 log. • The electrochemical treatment was performed at 0.9 V (vs. Ag/AgCl) for 15 min. • Electrochemical treatment caused oxidation of viral proteins. • Oxidation of viral proteins can lead to loss of viral structural integrity. - Abstract: Pathogenic viral infections are an international public health concern, and viral disinfection has received increasing attention. Electrochemical treatment has been used for treatment of water contaminated by bacteria for several decades, and although in recent years several reports have investigated viral inactivation kinetics, the mode of action of viral inactivation by electrochemical treatment remains unclear. Here, we demonstrated the inactivation of feline calicivirus (FCV), a surrogate for human noroviruses, by electrochemical treatment in a developed flow-cell equipped with a screen-printed electrode. The viral infectivity titer was reduced by over 5 orders of magnitude after 15 min of treatment at 0.9 V vs. Ag/AgCl. Proteomic study of electrochemically inactivated virus revealed oxidation of peptides located in the viral particles; oxidation was not observed in the non-treated sample. Furthermore, transmission electron microscopy revealed that viral particles in the treated sample had irregular structures. These results suggest that electrochemical treatment inactivates FCV via oxidation of peptides in the structural region, causing structural deformation of virus particles. This first report of viral protein damage through electrochemical treatment will contribute to broadening the understanding of viral inactivation mechanisms.

Quantum dot based on tin/titanium mixed oxide doped with europium synthesized by protein sol-gel method

International Nuclear Information System (INIS)

Paganini, Paula P.; Felinto, Maria Claudia F.C.; Brito, Hermi F.

2011-01-01

Special luminescence biomarkers have been developed to find more sensitive fluoroimmunoassay methods. A new generation of these biomarkers is the semiconductors nanocrystals, known as quantum dots, doped with lanthanides. The use of lanthanides ions as luminescent markers has many advantages, for example a security method, low cost, high specificity and also the luminescence can be promptly measured with high sensibility and accuracy. The protein sol-gel is a modification of conventional method, in which the coconut water replacing the alkoxides normally used. The advantage is that, the proteins present in coconut water bind chemically with metal salts forming a polymer chain. This work presents nanoparticles based on tin/titanium mixed oxide doped with 3% of europium synthesized by protein sol-gel method. The nanoparticles were burned at 300 deg C, 500 deg C, 800 deg C and 1100 deg C. The samples were analyzed and characterized by thermal analysis, X-ray powder diffraction (XRD), infrared spectroscopy (IR) and scanning electron microscopy (SEM). The synthesis was effective and the nanoparticles showed nanometric size and structural differences with the annealing. To be used in the fluoroimmunoassays tests, these particles need to be functionalized before be connect with biological molecules and after this process, these nanoparticles going to be submitted at gamma radiation for sterilization. (author)

Quantum dot based on tin/titanium mixed oxide doped with europium synthesized by protein sol-gel method

Energy Technology Data Exchange (ETDEWEB)

Paganini, Paula P.; Felinto, Maria Claudia F.C., E-mail: paulapaganini@usp.b, E-mail: mfelinto@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Brito, Hermi F., E-mail: hefbrito@iq.usp.b [Universidade de Sao Paulo (IQ/USP), Sao Paulo, SP (Brazil). Inst. de Quimica. Lab. de Elementos do Bloco f

2011-07-01

Special luminescence biomarkers have been developed to find more sensitive fluoroimmunoassay methods. A new generation of these biomarkers is the semiconductors nanocrystals, known as quantum dots, doped with lanthanides. The use of lanthanides ions as luminescent markers has many advantages, for example a security method, low cost, high specificity and also the luminescence can be promptly measured with high sensibility and accuracy. The protein sol-gel is a modification of conventional method, in which the coconut water replacing the alkoxides normally used. The advantage is that, the proteins present in coconut water bind chemically with metal salts forming a polymer chain. This work presents nanoparticles based on tin/titanium mixed oxide doped with 3% of europium synthesized by protein sol-gel method. The nanoparticles were burned at 300 deg C, 500 deg C, 800 deg C and 1100 deg C. The samples were analyzed and characterized by thermal analysis, X-ray powder diffraction (XRD), infrared spectroscopy (IR) and scanning electron microscopy (SEM). The synthesis was effective and the nanoparticles showed nanometric size and structural differences with the annealing. To be used in the fluoroimmunoassays tests, these particles need to be functionalized before be connect with biological molecules and after this process, these nanoparticles going to be submitted at gamma radiation for sterilization. (author)

Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

Directory of Open Access Journals (Sweden)

Allan Olspert

2016-06-01

Full Text Available Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV and murine norovirus (MNV. These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

Structural analysis of DNA–protein complexes regulating the restriction–modification system Esp1396I

International Nuclear Information System (INIS)

Martin, Richard N. A.; McGeehan, John E.; Ball, Neil J.; Streeter, Simon D.; Thresh, Sarah-Jane; Kneale, G. G.

2013-01-01

Comparison of bound and unbound DNA in protein–DNA co-crystal complexes reveals insights into controller-protein binding and DNA distortion in transcriptional regulation. The controller protein of the type II restriction–modification (RM) system Esp1396I binds to three distinct DNA operator sequences upstream of the methyltransferase and endonuclease genes in order to regulate their expression. Previous biophysical and crystallographic studies have shown molecular details of how the controller protein binds to the operator sites with very different affinities. Here, two protein–DNA co-crystal structures containing portions of unbound DNA from native operator sites are reported. The DNA in both complexes shows significant distortion in the region between the conserved symmetric sequences, similar to that of a DNA duplex when bound by the controller protein (C-protein), indicating that the naked DNA has an intrinsic tendency to bend when not bound to the C-protein. Moreover, the width of the major groove of the DNA adjacent to a bound C-protein dimer is observed to be significantly increased, supporting the idea that this DNA distortion contributes to the substantial cooperativity found when a second C-protein dimer binds to the operator to form the tetrameric repression complex

Protein carbonylation, protein aggregation and neuronal cell death in a murine model of multiple sclerosis

Science.gov (United States)

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

Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins

DEFF Research Database (Denmark)

Carroll, Luke; Pattison, David I; Davies, Justin B

2017-01-01

Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side-chain modification, backbone fragmentation, aggregation, and changes in structure...

Oxidative protein folding: from thiol-disulfide exchange reactions to the redox poise of the endoplasmic reticulum.

Science.gov (United States)

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.

Possibility of Modification of Zeolites by Iron Oxides and its Utilization for Removal of Pb(II from Water Solutions

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Michal Lovás

2004-12-01

Full Text Available Ion-exchange properties of cations from lattice and ions from solutions are characteristic for zeolites. Zeolites as sorbents are used in many branches of industry. Ion-exchange reactions of cations on zeolites have been a theme of many works. With the exception of using natural zeolites as the sorbent, a modification of surface of zeolites and preparation of synthetic zeolites has received interest lately. One of the common modification of zeolites is modification by iron oxides, which increases capacity of adsorption. In this work, we prepared a modified zeolite by the precipitation of magnetite on the surface of zeolite. This new adsorbent was used to remove of Pb(II from waste water. The maximum adsorption capacity was 73,25 mg/g from the solution of Pb with the concentration of 400 mg/l.

Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review

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Mina Ghahremani

2016-09-01

Full Text Available Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.

Oxidative Stress in the Carcinogenicity of Chemical Carcinogens

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Hideki Wanibuchi

2013-10-01

Full Text Available This review highlights several in vivo studies utilizing non-genotoxic and genotoxic chemical carcinogens, and the mechanisms of their high and low dose carcinogenicities with respect to formation of oxidative stress. Here, we survey the examples and discuss possible mechanisms of hormetic effects with cytochrome P450 inducers, such as phenobarbital, a-benzene hexachloride and 1,1-bis(p-chlorophenyl-2,2,2-trichloroethane. Epigenetic processes differentially can be affected by agents that impinge on oxidative DNA damage, repair, apoptosis, cell proliferation, intracellular communication and cell signaling. Non-genotoxic carcinogens may target nuclear receptors and induce post-translational modifications at the protein level, thereby impacting on the stability or activity of key regulatory proteins, including oncoproteins and tumor suppressor proteins. We further discuss role of oxidative stress focusing on the low dose carcinogenicities of several genotoxic carcinogens such as a hepatocarcinogen contained in seared fish and meat, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, arsenic and its metabolites, and the kidney carcinogen potassium bromate.

Oxidative stress-triggered interactions between the succinyl- and acetyl-proteomes of rice leaves.

Science.gov (United States)

Zhou, Heng; Finkemeier, Iris; Guan, Wenxue; Tossounian, Maria-Armineh; Wei, Bo; Young, David; Huang, Jingjing; Messens, Joris; Yang, Xibin; Zhu, Jun; Wilson, Michael H; Shen, Wenbiao; Xie, Yanjie; Foyer, Christine H

2018-05-01

Protein lysine acylations, such as succinylation and acetylation, are important post-translational modification (PTM) mechanisms, with key roles in cellular regulation. Antibody-based affinity enrichment, high-resolution liquid chromatography mass spectrometry analysis, and integrated bioinformatics analysis were used to characterize the lysine succinylome (K suc ) and acetylome (K ace ) of rice leaves. In total, 2,593 succinylated and 1,024 acetylated proteins were identified, of which 723 were simultaneously acetylated and succinylated. Proteins involved in photosynthetic carbon metabolism such as the large and small subunits of RuBisCO, ribosomal functions, and other key processes were subject to both PTMs. Preliminary insights into oxidant-induced changes to the rice acetylome and succinylome were gained from treatments with hydrogen peroxide. Exposure to oxidative stress did not regulate global changes in the rice acetylome or succinylome but rather led to modifications on a specific subset of the identified sites. De-succinylation of recombinant catalase (CATA) and glutathione S-transferase (OsGSTU6) altered the activities of these enzymes showing that this PTM may have a regulatory function. These findings not only greatly extend the list of acetylated and/or succinylated proteins but they also demonstrate the close cooperation between these PTMs in leaf proteins with key metabolic functions. © 2017 John Wiley & Sons Ltd.

Conflict RNA modification, host-parasite co-evolution, and the origins of DNA and DNA-binding proteins1.

Science.gov (United States)

McLaughlin, Paul J; Keegan, Liam P

2014-08-01

Nearly 150 different enzymatically modified forms of the four canonical residues in RNA have been identified. For instance, enzymes of the ADAR (adenosine deaminase acting on RNA) family convert adenosine residues into inosine in cellular dsRNAs. Recent findings show that DNA endonuclease V enzymes have undergone an evolutionary transition from cleaving 3' to deoxyinosine in DNA and ssDNA to cleaving 3' to inosine in dsRNA and ssRNA in humans. Recent work on dsRNA-binding domains of ADARs and other proteins also shows that a degree of sequence specificity is achieved by direct readout in the minor groove. However, the level of sequence specificity observed is much less than that of DNA major groove-binding helix-turn-helix proteins. We suggest that the evolution of DNA-binding proteins following the RNA to DNA genome transition represents the major advantage that DNA genomes have over RNA genomes. We propose that a hypothetical RNA modification, a RRAR (ribose reductase acting on genomic dsRNA) produced the first stretches of DNA in RNA genomes. We discuss why this is the most satisfactory explanation for the origin of DNA. The evolution of this RNA modification and later steps to DNA genomes are likely to have been driven by cellular genome co-evolution with viruses and intragenomic parasites. RNA modifications continue to be involved in host-virus conflicts; in vertebrates, edited cellular dsRNAs with inosine-uracil base pairs appear to be recognized as self RNA and to suppress activation of innate immune sensors that detect viral dsRNA.

Effect of pasteurization on the protein composition and oxidative stability of beer during storage.

Science.gov (United States)

Lund, Marianne N; Hoff, Signe; Berner, Torben S; Lametsch, René; Andersen, Mogens L

2012-12-19

The impacts of pasteurization of a lager beer on protein composition and the oxidative stability were studied during storage at 22 °C for 426 days in the dark. Pasteurization clearly improved the oxidative stability of beer determined by ESR spectroscopy, whereas it had a minor negative effect on the volatile profile by increasing volatile compounds that is generally associated with heat treatment and a loss of fruity ester aroma. A faster rate of radical formation in unpasteurized beer was consistent with a faster consumption of sulfite. Beer proteins in the unpasteurized beer were more degraded, most likely due to proteolytic enzyme activity of yeast remnants and more precipitation of proteins was also observed. The differences in soluble protein content and composition are suggested to result in differences in the contents of prooxidative metals as a consequence of the proteins ability to bind metals. This also contributes to the differences in oxidative stabilities of the beers.

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

Energy Technology Data Exchange (ETDEWEB)

Lee, Minjung [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine and Samsung Biomedical Research Institute, Suwon-Si, Kyonggi-Do (Korea, Republic of); Shin, Jaekyoon, E-mail: jkshin@med.skku.ac.kr [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine and Samsung Biomedical Research Institute, Suwon-Si, Kyonggi-Do (Korea, Republic of)

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. 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.

Oxidative Modification of Tryptophan-Containing Peptides

DEFF Research Database (Denmark)

Petersen, Jonas; Christensen, Pia Katrine; Nielsen, Mathias T

2018-01-01

We herein present a broadly useful method for the chemoselective modification of a wide range of tryptophan-containing peptides. Exposing a tryptophan-containing peptide to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) resulted in a selective cyclodehydration between the peptide backbone...

The Role of Protein Modifications of T-Bet in Cytokine Production and Differentiation of T Helper Cells

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Sera Oh

2014-01-01

Full Text Available T-Bet (T-box protein expressed in T cells, also called as TBX21 was originally cloned as a key transcription factor involved in the commitment of T helper (Th cells to the Th1 lineage. T-Bet directly activates IFN-γ gene transcription and enhances development of Th1 cells. T-Bet simultaneously modulates IL-2 and Th2 cytokines in an IFN-γ-independent manner, resulting in an attenuation of Th2 cell development. Numerous studies have demonstrated that T-bet plays multiple roles in many subtypes of immune cells, including B cell, dendritic cells, natural killer (NK cells, NK T cells, and innate lymphoid cells. Therefore, T-bet is crucial for the development and coordination of both innate and adaptive immune responses. To fulfill these multiple roles, T-bet undergoes several posttranslational protein modifications, such as phosphorylation at tyrosine, serine, and threonine residues, and ubiquitination at lysine residues, which affect lineage commitment during Th cell differentiation. This review presents a current overview of the progress made in understanding the roles of various types of T-bet protein modifications in the regulation of cytokine production during Th cell differentiation.

Tandem Affinity Purification Approach Coupled to Mass Spectrometry to Identify Post-translational Modifications of Histones Associated with Chromatin-Binding Proteins.

Science.gov (United States)

Beyer, Sophie; Robin, Philippe; Ait-Si-Ali, Slimane

2017-01-01

Protein purification by tandem affinity purification (TAP)-tag coupled to mass spectrometry analysis is usually used to reveal protein complex composition. Here we describe a TAP-tag purification of chromatin-bound proteins along with associated nucleosomes, which allow exhaustive identification of protein partners. Moreover, this method allows exhaustive identification of the post-translational modifications (PTMs) of the associated histones. Thus, in addition to partner characterization, this approach reveals the associated epigenetic landscape that can shed light on the function and properties of the studied chromatin-bound protein.

A directional entrapment modification on the polyethylene surface by the amphiphilic modifier of stearyl-alcohol poly(ethylene oxide) ether

Science.gov (United States)

Lu, Qiang; Chen, Yi; Huang, Juexin; Huang, Jian; Wang, Xiaolin; Yao, Jiaying

2018-05-01

A novel entrapment modification method involving directional implantation of the amphiphilic modifier of stearyl-alcohol poly(ethylene oxide) ether (AEO) into the high-density polyethylene (HDPE) surface is proposed. This modification technique allows the AEO modifier to be able to spontaneously attain and subsequently penetrate into the swollen HDPE surface with its hydrophobic stearyl segment, while its hydrophilic poly(ethylene oxide) (PEO) segment spontaneously points to water. The AEO modifier with a HLB number below 8.7 was proved appropriate for the directional entrapment, Nevertheless, AEOs with larger HLB numbers were also effective modifiers in the presence of salt additives. In addition, a larger and hydrophobic micelle, induced respectively by the AEO concentration above 1.3 × 10-2 mol/L and the entrapping temperature above the cloud point of AEO, could lead to a sharp contact angle decline of the modified surface. Finally, a hydrophilic HDPE surface with the modifier coverage of 38.9% was reached by the directional entrapment method, which is far larger than that of 19.2% by the traditional entrapment method.

Modification-specific proteomics: strategies for characterization of post-translational modifications using enrichment techniques

DEFF Research Database (Denmark)

Zhao, Yingming; Jensen, Ole N

2009-01-01

More than 300 different types of protein post-translational modifications (PTMs) have been described, many of which are known to have pivotal roles in cellular physiology and disease. Nevertheless, only a handful of PTMs have been extensively investigated at the proteome level. Knowledge of protein...... substrates and their PTM sites is key to dissection of PTM-mediated cellular processes. The past several years have seen a tremendous progress in developing MS-based proteomics technologies for global PTM analysis, including numerous studies of yeast and other microbes. Modification-specific enrichment...

Chemical Modifications of Starch: Microwave Effect

OpenAIRE

Lewicka, Kamila; Siemion, Przemysław; Kurcok, Piotr

2015-01-01

This paper presents basic methods of starch chemical modification, the effect of microwave radiation on the modification process, and the physicochemical properties of starch. It has been shown that the modifications contribute to improvement of the material performance and likewise to significant improvement of its mechanical properties. As a result, more and more extensive use of starch is possible in various industries. In addition, methods of oxidized starch and starch esters preparation ...

Serum Advanced Oxidation Protein Products in Oral Squamous Cell Carcinoma: Possible Markers of Diagnostic Significance

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Abhishek Singh Nayyar

2013-07-01

Full Text Available Background: The aim of this study was to measure the concentrations (levels ofserum total proteins and advanced oxidation protein products as markers of oxidantmediated protein damage in the sera of patients with oral cancers.Methods: The study consisted of the sera analyses of serum total protein andadvanced oxidation protein products’ levels in 30 age and sex matched controls, 60patients with reported pre-cancerous lesions and/or conditions and 60 patients withhistologically proven oral squamous cell carcinoma. One way analyses of variance wereused to test the difference between groups. To determine which of the two groups’ meanswere significantly different, the post-hoc test of Bonferroni was used. The results wereaveraged as mean ± standard deviation. In the above test, P values less than 0.05 weretaken to be statistically significant. The normality of data was checked before thestatistical analysis was performed.Results: The study revealed statistically significant variations in serum levels ofadvanced oxidation protein products (P<0.001. Serum levels of total protein showedextensive variations; therefore the results were largely inconclusive and statisticallyinsignificant.Conclusion: The results emphasize the need for more studies with larger samplesizes to be conducted before a conclusive role can be determined for sera levels of totalprotein and advanced oxidation protein products as markers both for diagnosticsignificance and the transition from the various oral pre-cancerous lesions and conditionsinto frank oral cancers.

Glycation and glycoxidation of low-density lipoproteins by glucose and low-molecular mass aldehydes. Formation of modified and oxidized particles

DEFF Research Database (Denmark)

Knott, Heather M; Brown, Bronwyn E; Davies, Michael Jonathan

2003-01-01

of macrophages. This results in the accumulation of cholesterol and cholesteryl esters within macrophages and the formation of foam cells, a hallmark of atherosclerosis. The nature of the LDL modifications required for cellular recognition and unregulated uptake are poorly understood. We have therefore examined...... or methylglyoxal in the presence of copper ions, whereas glycolaldehyde stimulated such reactions to a modest extent. These results suggest that the earliest significant events in this system are metal ion-independent glycation (modification) of the protein component of LDL, whilst oxidative events (glycoxidation...... or direct oxidation of lipid or proteins) only occur to any significant extent at later time points. This 'carbonyl-stress' may facilitate the formation of foam cells and the vascular complications of diabetes....

Protein aggregation in food models: effect of γ-irradiation and lipid oxidation

International Nuclear Information System (INIS)

Delincee, H.; Paul, P.

1981-01-01

Myoglobin and serum albumin have been irradiated in aqueous solution in the presence of varying amounts of carbohydrates and lipids, and the yield of protein aggregates has been determined by gel filtration. With myoglobin the formation of aggregates evolving from the reaction with oxidizing lipids was observed, which was not found for serum albumin. The production of protein-lipid complexes, in which lipid material was occluded in the high-molecular aggregates by physical forces was demonstrated. Gel filtration and gel electrophoresis, both in the presence of SDS, and thin-layer isoelectric focusing revealed distinct structural differenes between the protein aggregates induced by irradiation and the aggregates formed by interaction with oxidizing lipids

Pengaruh Penggunaan Formalin terhadap Kerusakan Protein Daging Ikan Tuna (Thunus sp)

OpenAIRE

Apituley, Daniel A. N.

2012-01-01

Formaldehyde is used on handling and processing of fishery product by some fisher at some coastal region of Indonesia was one that promotes protein oxidation and modification of protein structure of fish meat which give direct impact on nutritional quality of fish primarily the amino acids. This experiment was done in vitro experiment by using model of protein preparat of red and white meat of tuna. The purpose of this research was to study the effect of formaldehyde concentration and impact ...

γ-Glutamyl semialdehyde and 2-amino-adipic semialdehyde: biomarkers of oxidative damage to proteins

DEFF Research Database (Denmark)

Daneshvar, B.; Frandsen, H.; Autrup, Herman

1997-01-01

proteins collected from eight different mammalian species was found to be inversely proportional to their maximum lifespan potential. The content of AAS in plasma proteins of untreated adult rats showed a positive correlation with the age of the rat. In young rats a negative correlation with age was found......Reactive oxygen species are formed in the body by several natural processes and by induced oxidative stress. The reactive oxygen species may react with the various biomolecules of the body, including proteins. In order to assess the impact of oxidative damage to proteins, we have tried to identify...

Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division

KAUST Repository

An, Xiaomeng

2017-06-21

Caenorhabditis briggsae has emerged as a model for comparative biology against model organism C. elegans. Most of its cell fate specifications are completed during embryogenesis whereas its cell growth is achieved mainly in larval stages. The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins in the C. briggsae embryos and larvae, 247 of which undergo up- or down-regulation between the two stages. The proteins that are upregulated in the larval stages are enriched in the Gene Ontology categories of energy production, protein translation, and cytoskeleton; whereas those upregulated in the embryonic stage are enriched in the categories of chromatin dynamics and posttranslational modification, suggesting a more active chromatin modification in the embryos than in the larva. Perturbation of a subset of chromatin modifiers followed by cell lineage analysis suggests their roles in controlling cell division pace. Taken together, we demonstrate a general molecular switch from chromatin modification to metabolism during the transition from C. briggsae embryonic to its larval stages using iTRAQ approach. The switch might be conserved across metazoans.

4-Hydroxyhexenal- and 4-Hydroxynonenal-Modified Proteins in Pterygia

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Ichiya Sano

2013-01-01

Full Text Available Oxidative stress has been suspected of contributing to the pathogenesis of pterygia. We evaluated the immunohistochemical localization of the markers of oxidative stress, that is, the proteins modified by 4-hydroxyhexenal (4-HHE and 4-hydroxynonenal (4-HNE, which are reactive aldehydes derived from nonenzymatic oxidation of n-3 and n-6 polyunsaturated fatty acids, respectively. In the pterygial head, labeling of 4-HHE- and 4-HNE-modified proteins was prominent in the nuclei and cytosol of the epithelium. In the pterygial body, strong labeling was observed in the nuclei and cytosol of the epithelium and proliferating subepithelial connective tissue. In normal conjunctival specimens, only trace immunoreactivity of both proteins was observed in the epithelial and stromal layers. Exposures of ultraviolet (330 nm, 48.32 ± 0.55 J/cm2 or blue light (400 nm, 293.0 ± 2.0 J/cm2 to rat eyes enhanced labeling of 4-HHE- and 4-HNE-modified proteins in the nuclei of conjunctival epithelium. Protein modifications by biologically active aldehydes are a molecular event involved in the development of pterygia.

Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors

DEFF Research Database (Denmark)

Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

2015-01-01

taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we...

Comparison of Oxidative Stresses Mediated by Different Crystalline Forms and Surface Modification of Titanium Dioxide Nanoparticles

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Karim Samy El-Said

2015-01-01

Full Text Available Titanium dioxide nanoparticles (TiO2 NPs are manufactured worldwide for use in a wide range of applications. There are two common crystalline forms of TiO2 anatase and rutile with different physical and chemical characteristics. We previously demonstrated that an increased DNA damage response is mediated by anatase crystalline form compared to rutile. In the present study, we conjugated TiO2 NPs with polyethylene glycol (PEG in order to reduce the genotoxicity and we evaluated some oxidative stress parameters to obtain information on the cellular mechanisms of DNA damage that operate in response to TiO2 NPs different crystalline forms exposure in hepatocarcinoma cell lines (HepG2. Our results indicated a significant increase in oxidative stress mediated by the anatase form of TiO2 NPs compared to rutile form. On the other hand, PEG modified TiO2 NPs showed a significant decrease in oxidative stress as compared to TiO2 NPs. These data suggested that the genotoxic potential of TiO2 NPs varies with crystalline form and surface modification.

Antigen retrieval prior to on-tissue digestion of formalin-fixed paraffin-embedded tumour tissue sections yields oxidation of proline residues.

Science.gov (United States)

Djidja, Marie-Claude; Claude, Emmanuelle; Scriven, Peter; Allen, David W; Carolan, Vikki A; Clench, Malcolm R

2017-07-01

MALDI-mass spectrometry imaging (MALDI-MSI) has been shown to allow the study of protein distribution and identification directly within formalin-fixed paraffin-embedded (FFPE) tissue sections. However, direct protein identification from tissue sections remains challenging due to signal interferences and/or existing post-translational or other chemical modifications. The use of antigen retrieval (AR) has been demonstrated for unlocking proteins prior to in situ enzymatic digestion and MALDI-MSI analysis of FFPE tissue sections. In the work reported here, the identification of proline oxidation, which may occur when performing the AR protocol, is described. This facilitated and considerably increased the number of identified peptides when adding proline oxidation as a variable modification to the MASCOT search criteria. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

Possible involvement of GABAergic mechanism in protective effect of melatonin against sleep deprivation-induced behaviour modification and oxidative damage in mice.

Science.gov (United States)

Kumar, Anil; Singh, Anant

2009-08-01

Sleep is an important physiological process responsible for the maintenance of physical, mental and emotional health of a living being. Sleep deprivation is considered risky for several pathological diseases such as anxiety and motor and cognitive dysfunctions. Sleep deprivation has recently been reported to cause oxidative damage. This study has been designed to explore the possible involvement of the GABAergic mechanism in protective effects of melatonin against 72-h sleep deprivation-induced behaviour modification and oxidative damage in mice. Mice were sleep-deprived for a period of 72 h using the grid over water suspended method. Animals were divided into groups of 6-8 animals each. Melatonin (5 and 10 mg/kg), flumazenil (0.5 mg/kg), picrotoxin (0.5 mg/kg) and muscimol (0.05 mg/kg) were administered for 5 days starting 2 days before 72-h sleep deprivation. Various behavioural tests (plus maze, zero maze, mirror chamber, actophotometer) and body weight assessment followed by oxidative stress parameters (malondialdehyde level, glutathione, catalase, nitrite and protein) were carried out. The 72-h sleep deprivation caused significant anxiety-like behaviour, weight loss, impaired locomotor activity and oxidative damage as compared with naïve (without sleep deprivation). Treatment with melatonin (5 mg/kg and 10 mg/kg, ip) significantly improved locomotor activity, weight loss and antianxiety effect as compared with control (sleep-deprived). Biochemically, melatonin treatment significantly restored reduced glutathione, catalase activity, attenuated lipid peroxidation and nitrite level as compared with control animals (72-h sleep-deprived). Flumazenil (0.5 mg/kg) and picrotoxin (0.5 mg/kg) pretreatments with a lower dose of melatonin (5 mg/kg) significantly antagonized the protective effect of melatonin. However, muscimol (0.05 mg/kg) pretreatment with melatonin (5 mg/kg, ip) potentiated the protective effect of melatonin which was significant as compared with their

Glycation and glycoxidation of low-density lipoproteins by glucose and low-molecular mass aldehydes. Formation of modified and oxidized particles

DEFF Research Database (Denmark)

Knott, Heather M; Brown, Bronwyn E; Davies, Michael Jonathan

2003-01-01

by the adduction of glucose or species derived from glucose, such as low-molecular mass aldehydes, to proteins. These reactions can be nonoxidative (glycation) or oxidative (glycoxidation) and result in the conversion of low-density lipoproteins (LDL) to a form that is recognized by the scavenger receptors...... with glucose. These processes are rapid and unaffected by low concentrations of copper ions. In contrast, lipid and protein oxidation are slow processes and occur to a limited extent in the absence of added copper ions. No evidence was obtained for the stimulation of lipid or protein oxidation by glucose...... or methylglyoxal in the presence of copper ions, whereas glycolaldehyde stimulated such reactions to a modest extent. These results suggest that the earliest significant events in this system are metal ion-independent glycation (modification) of the protein component of LDL, whilst oxidative events (glycoxidation...

Improved efficiency of nanoneedle insertion by modification with a cell-puncturing protein

Science.gov (United States)

Ryu, Seunghwan; Matsumoto, Yuta; Matsumoto, Takahiro; Ueno, Takafumi; Silberberg, Yaron R.; Nakamura, Chikashi

2018-03-01

An atomic force microscope (AFM) probe etched into an ultra-sharp cylindrical shape (a nanoneedle) can be inserted into a living cell and mechanical responses of the insertion process are represented as force-distance curves using AFM. A probe-molecule-functionalized nanoneedle can be used to detect intracellular molecules of interest in situ. The insertion efficiencies of nanoneedles vary among cell types due to the cortex structures of cells, and some cell types, such as mouse fibroblast Balb/3T3 cells, show extremely low efficacy of insertion. We addressed this issue by using a cell membrane puncturing protein from bacteriophage T4 (gp5), a needle-like protein that spontaneously penetrates through the cell membrane. Gp5 was immobilized onto a nanoneedle surface. The insertion efficiency of the functionalized nanoneedle increased by over 15% compared to the non-functionalized control. Gp5-modification is a versatile approach in cell manipulation techniques for the insertion of other types of nanostructures into cells.

In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae.

Science.gov (United States)

Kurotani, Atsushi; Sakurai, Tetsuya

2015-08-20

Recent proteome analyses have reported that intrinsically disordered regions (IDRs) of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs) has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST) and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups.

In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae

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Atsushi Kurotani

2015-08-01

Full Text Available Recent proteome analyses have reported that intrinsically disordered regions (IDRs of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups.