Interaction of reactive oxygen and nitrogen species with proteins

Free radicals and reactive oxygen or nitrogen species generated during oxidative stress and as by-products of normal cellular metabolism may damage all types of biological molecules. Proteins are major initial targets in cell. Reactions of a variety of free radicals and reactive oxygen and nitrogen species with proteins can lead to oxidative modifications of proteins such as protein hydroperoxides formation, hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, oxidation of sulfhydryl groups, oxidation of methionine residues, conversion of some amino acid residues into carbonyl groups, cleavage of the polypeptide chain and formation of cross-linking bonds. Such modifications of proteins leading to loss of their function (enzymatic activity), accumulation and inhibition of their degradation have been observed in several human diseases, aging, cell differentiation and apoptosis. Formation of specific protein oxidation products may be used as biomarkers of oxidative stress.     

Permeability of milk protein antigens across the intestinal epithelium in vitro

Degradations by proteolytic enzymes and intestinal epithelial permeability represent two major drawbacks to the transfer of food protein antigens to blood. These steps were studied in vitro for the milk protein antigens beta-lactoglobulin (beta-Lg), alpha-Lactalbumin (alpha-La) and beta-casein (beta-cas). Pepsin-trypsin hydrolysis and permeability in isolated rabbit ileum in Ussing chamber were suited by ELISA and radiolabelled-protein measurement. Pepsin-trypsin hydrolysis showed an increasing resistance in the order beta-cas less than alpha-La less than beta-Lg. The rate of absorption of the antigenic proteins by isolated rabbit ileum was in the same order, and the rate of absorption of the whole proteins (degraded and antigenic forms) was significantly higher for beta-Lg than for alpha-La and beta-cas. These results suggest a selective intestinal permeability for milk protein antigens. This selectivity is probably important in the mechanism of food protein sensitization via the oral route.     

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier.     

Site-selective modifications of arginine residues in human hemoglobin induced by methylglyoxal

Methylglyoxal (MG) is an important glycating agent produced under physiological conditions. MG could react with DNA and proteins to generate advanced glycation end products. Human hemoglobin, the most abundant protein in blood cells, has not been systematically investigated as the target protein for methylglyoxal modification. Here we examined carefully, by using HPLC coupled with tandem mass spectrometry (LC-MS/MS), the covalent modifications of human hemoglobin induced by methylglyoxal. Our results revealed that hemoglobin could be modified by methylglyoxal, and the major form of modification was found to be the hydroimidazolone derivative of arginine residues. In addition, Arg-92 and Arg-141 in the alpha chain as well as Arg-40 and Arg-104 in the beta chain were modified, whereas two other arginine residues, that is, Arg-31 in the alpha chain and Arg-30 in the beta chain, were not modified. Semiquantitative measurement for adduct formation, together with the analysis of the X-ray structure of hemoglobin, showed that the extents of arginine modification were highly correlated with the solvent accessibilities of these residues. The facile formation of hydroimidazolone derivatives of arginine residues in hemoglobin by methylglyoxal at physiologically relevant concentrations suggested that this type of modification might occur in vivo. The unambiguous determination of the sites and extents of methylglyoxal modifications of arginines in hemoglobin provided a basis for understanding the implications of these modifications and for employing this type of hemoglobin modification as molecular biomarkers for clinical applications.     

Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications

Filamentous inclusions of alpha-synuclein protein are hallmarks of neurodegenerative diseases collectively known as synucleinopathies. Previous studies have shown that exposure to oxidative and nitrative species stabilizes alpha-synuclein filaments in vitro, and this stabilization may be due to dityrosine cross-linking. To test this hypothesis, we mutated tyrosine residues to phenylalanine and generated recombinant wild type and mutant alpha-synuclein proteins. alpha-Synuclein proteins lacking some or all tyrosine residues form fibrils to the same extent as the wild type protein. Tyrosine residues are not required for protein cross-linking or filament stabilization resulting from transition metal-mediated oxidation, because higher Mr SDS-resistant oligomers and filaments stable to chaotropic agents are detected using all Tyr --> Phe alpha-synuclein mutants. By contrast, cross-linking resulting from exposure to nitrating agents required the presence of one or more tyrosine residues. Furthermore, tyrosine cross-linking is involved in filament stabilization, because nitrating agent-exposed assembled wild type, but not mutant alpha-synuclein lacking all tyrosine residues, was stable to chaotropic treatment. In addition, the formation of stable alpha-synuclein inclusions in intact cells after exposure to oxidizing and nitrating species requires tyrosine residues. These findings demonstrate that nitrative and/or oxidative stress results in distinct mechanisms of alpha-synuclein protein modifications that can influence the formation of stable alpha-synuclein fibrils.     

Biological selectivity and functional aspects of protein tyrosine nitration

The formation of nitric oxide in biological systems has led to the discovery of a number of post-translational protein modifications that could regulate protein function or potentially be utilized as transducers of nitric oxide signaling. Principal among the nitric oxide-mediated protein modifications are: the nitric oxide-iron heme binding, the S-nitrosylation of reduced cysteine residues, and the C-nitration of tyrosine and tryptophan residues. With the exception of the nitric oxide binding to heme iron proteins, the other two modifications appear to require secondary reactions of nitric oxide and the formation of nitrogen oxides. The rapid development of analytical and immunological methodologies has allowed for the quantification of S-nitrosylated and C-nitrated proteins in vivo revealing an apparent selectivity and specificity of the proteins modified. This review is primarily focused upon the nitration of tyrosine residues discussing parameters that may govern the in vivo selectivity of protein nitration, and the potential biological significance and clinical relevance of this nitric oxide-mediated protein modification.     

Immunogenicity of homologous low density lipoprotein after methylation, ethylation, acetylation, or carbamylation: generation of antibodies specific for derivatized lysine

We previously showed that immunization of guinea pigs with reductively glucosylated guinea pig low density lipoprotein (LDL) or albumin resulted in the formation of antibodies specific for the glucosylated protein. The present studies were done to determine if modifications of homologous LDL or albumin, other than addition of carbohydrate, would also render these proteins immunogenic. We found that derivatization of lysine residues of guinea pig LDL or albumin by carbamylation, acetylation, ethylation, or even methylation rendered them immunogenic in guinea pigs. In addition, the specificity of the antibodies was strikingly influenced by whether modified homologous LDL or modified homologous albumin was used as the immunogen. Antibodies generated against modified LDL were directed almost exclusively against the derivatized lysine residues (i.e., carbamyllysine, acetyllysine, or methyllysine) and hence reacted equivalently with other modified proteins that contained the same lysine derivative. However, antibodies generated against guinea pig albumin (or fibrinogen) modified in the same ways reacted primarily with the modified protein used as immunogen, and not with the free lysine derivative, or with other similarly modified proteins. Each of the modifications referred to above could potentially occur in vivo. Therefore, the findings presented may be relevant to autoantibody formation and immunopathogenetic mechanisms in certain diseases.     

Increased methyl esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative stress.

Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT; EC 2. 1.1.77) catalyses the methyl esterification of the free alpha-carboxyl group of abnormal L-isoaspartyl residues, which occur spontaneously in protein and peptide substrates as a consequence of molecular ageing. The biological function of this transmethylation reaction is related to the repair or degradation of age-damaged proteins. Methyl ester formation in erythrocyte membrane proteins has also been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases, such as hereditary spherocytosis, or cell stress (thermal or osmotic) conditions. The study shows that levels of L-isoaspartyl residues rise in membrane proteins of human erythrocytes exposed to oxidative stress, induced by t-butyl hydroperoxide or H2O2. The increase in malondialdehyde content confirmed that the cell membrane is a primary target of oxidative alterations. A parallel rise in the methaemoglobin content indicates that proteins are heavily affected by the molecular alterations induced by oxidative treatments in erythrocytes. Antioxidants largely prevented the increase in membrane protein methylation, underscoring the specificity of the effect. Conversely, we found that PCMT activity, consistent with its repair function, remained remarkably stable under oxidative conditions, while damaged membrane protein substrates increased significantly. The latter include ankyrin, band 4.1 and 4.2, and the integral membrane protein band 3 (the anion exchanger). The main target was found to be particularly protein 4.1, a crucial element in the maintenance of membrane-cytoskeleton network stability. We conclude that the increased formation/exposure of L-isoaspartyl residues is one of the major structural alterations occurring in erythrocyte membrane proteins as a result of an oxidative stress event. In the light of these and previous findings, the occurrence of isoaspartyl sites in membrane proteins as a key event in erythrocyte spleen conditioning and hemocatheresis is proposed.     

Utilization of fluorescent probes for the quantification and identification of subcellular proteomes and biological processes regulated by lipid peroxidation products

Oxidative modifications to cellular proteins are critical in mediating redox-sensitive processes such as autophagy, the antioxidant response, and apoptosis. The proteins that become modified by reactive species are often compartmentalized to specific organelles or regions of the cell. Here, we detail protocols for identifying the subcellular protein targets of lipid oxidation and for linking protein modifications with biological responses such as autophagy. Fluorophores such as BODIPY-labeled arachidonic acid or BODIPY-conjugated electrophiles can be paired with organelle-specific probes to identify specific biological processes and signaling pathways activated in response to oxidative stress. In particular, we demonstrate “negative” and “positive” labeling methods using BODIPY-tagged reagents for examining oxidative modifications to protein nucleophiles. The protocol describes the use of these probes in slot immunoblotting, quantitative Western blotting, in-gel fluorescence, and confocal microscopy techniques. In particular, the use of the BODIPY fluorophore with organelle- or biological process-specific dyes and chromophores is highlighted. These methods can be used in multiple cell types as well as isolated organelles to interrogate the role of oxidative modifications in regulating biological responses to oxidative stress.     

Fluorescent and affinity-based tools to detect cysteine sulfenic acid formation in proteins

Cysteine sulfenic acid formation in proteins results from the oxidative modification of susceptible cysteine residues by hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This species represents a biologically significant modification occurring during oxidant signaling or oxidative stress, and it can modulate protein function. Most methods to identify such oxidatively modified proteins rely on monitoring the loss of one or more thiol group(s) or on selective labeling of nascent thiol groups following reduction of oxidized proteins. Our previous work reported the direct labeling of these chemically distinct modifications with a dimedone analogue, 1,3-cyclohexadione, to which a linker and functional group (an alcohol) had been added; further addition of a fluorescent isatoic acid or methoxycoumarin reporter allowed detection of the incorporated tag by fluorescence techniques ( Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M., and King, S. B. ( 2005) Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjugate Chem . 16, 1624-1628 ). We have now expanded our arsenal of tagging reagents to include two fluorescein-, two rhodamine-, and three biotin-conjugated probes based on the original approach. The new tools provide readily detectable fluorescent and affinity probes to identify sulfenic acid modifications in proteins and have been used in subsequent mass spectrometric analyses to confirm covalent attachment of the conjugates and directly determine the site of modification.     

Posttranslational modification of human alphaA-crystallin: correlation with electrophoretic migration.

alphaA-crystallin is a major protein component of the human lens. It is known to undergo posttranslational modification. This study was done to further elucidate the temporal and spatial nature of these posttranslational modifications and to correlate the modified forms with electrophoretic migration. We dissected normal human lenses into concentric shells of fiber cells, separated the proteins by two-dimensional electrophoresis, and identified modified forms by mass spectrometry. We found that alphaA-crystallin migrated as a major spot and in over 20 additional protein spots. The extent of modification correlated with the age of the fiber cells and the depth within a lens. A correlation was also seen between these parameters and the concentration of modified forms that had full-length sequences but migrated at more acidic positions. These proteins were phosphorylated, acetylated, and/or deamidated. A few proteins migrated to a more basic position than the major form of alphaA-crystallin. The locations of several species that were truncated after C-terminal residues Ser172 and Ser162 were identified. Each of these species had intact N termini. The similarity of the C-terminal cleavage sites found in alphaA- and alphaB-crystallins was noted.     

The ligand-binding site of bovine beta-lactoglobulin: evidence for a function?

Ever since the fortuitous observation that beta-lactoglobulin (beta-Lg), the major whey protein in the milk of ruminants, bound retinol, the details of the binding have been controversial. beta-Lg is a lipocalin, like plasma retinol-binding protein, so that ligand association was expected to make use of the central cavity in the protein. However, an early crystallographic analysis and some of the more recent solution studies indicated binding elsewhere. We have now determined the crystal structures of the complexes of the trigonal form of beta-Lg at pH 7.5 with bound retinol (R=21.4% for 7329 reflections between 20 and 2.4 A resolution, R(free)=30.6%) and with bound retinoic acid (R=22.7% for 7813 reflections between 20 and 2.34 A resolution, R(free)=29.8%). Both ligands are found to occupy the central calyx in a manner similar to retinol binding in retinol-binding protein. We find no evidence of binding at the putative external binding site in either of these structural analyses. Further, competition between palmitic acid and retinol reveals only palmitate bound to the protein. An explanation is provided for the lack of ligand binding to the orthorhombic crystal form also obtained at pH 7.5. Finally, the possible function of beta-Lg is discussed in the light of its species distribution and similarity to other lipocalins.     

Oxidative Damage of Lysozyme and Human Serum Albumin and Their Mixtures. A Comparison of Photosensitized and Peroxyl Radical Promoted Processes

Oxidative modifications of lysozyme (Lyso) and human serum albumin (HSA) mediated by photoinduced processes and peroxyl radicals were studied. Both oxidative conditions were applied to the separate proteins and their mixtures. Dimerization and fragmentation of the proteins do not correlate with the formation of carbonyls or peroxides, implying that evaluation of these changes is not an index of the overall oxidative modification of a protein. The results obtained also show that the hypothesis that the electrostatic interactions of Lyso and HSA could facilitate the formation of Lyso-HSA dimers in the presence of a source of reactive oxygen species was verified in both ROS-producing systems.     

Structure and function of proteins involved in milk allergies

Allergy to milk proteins has been defined as any adverse reaction mediated by immunological mechanisms to one or several of proteins found in milk. The milk allergy has been classified according to the onset of symptoms as immediate or delayed type. The milk allergy seems to be manifested by three major proteins found in milk: α-lactalbumin, β-lactoglobulin and caseins. The structural comparison of allergenic sites in α-lactalbumin and β-lactoglobulin with the structure of lactoferrin has clearly shown that yet another major milk protein lactoferrin also possesses allergenic sites and thus may qualify to be an allergen. The heat treatment of milk proteins considerably reduces their allergenicity.     

Oxidant Sensing by Reversible Disulfide Bond Formation

Maintenance of the cellular redox balance is crucial for cell survival. An increase in reactive oxygen, nitrogen, or chlorine species can lead to oxidative stress conditions, potentially damaging DNA, lipids, and proteins. Proteins are very sensitive to oxidative modifications, particularly methionine and cysteine residues. The reversibility of some of these oxidative protein modifications makes them ideally suited to take on regulatory roles in protein function. This is especially true for disulfide bond formation, which has the potential to mediate extensive yet fully reversible structural and functional changes, rapidly adjusting the protein''s activity to the prevailing oxidant levels.     

Oxidative stresses induced by glycoxidized human or bovine serum albumin on human monocytes

Oxidative stress and protein modifications are frequently observed in numerous disease states. Albumin, the major circulating protein in blood, can undergo increased glycoxidation in diabetes. Protein glycoxidation can lead to the formation of advanced glycoxidation end products, which induce various deleterious effects on cells. Herein, we report the effect of glucose or methylglyoxal-induced oxidative modifications on BSA or HSA protein structures and on THP1 monocyte physiology. The occurrence of oxidative modifications was found to be enhanced in glycoxidized BSA and HSA, after determination of their free thiol group content, relative electrophoretic migration, carbonyl content, and antioxidant activities. Cells treated with glycoxidized albumin exhibited an overgeneration of intracellular reactive oxygen species, impairments in proteasomal activities, enhancements in RAGE expression, and an accumulation of carbonylated proteins. These novel observations made in the presence of a range of modified BSA and HSA facilitate the comparison of the glycoxidation extent of albumin with the oxidative stress induced in cultured monocytes. Finally, this study reconfirms the influence of experimental conditions in which AGEs are generated and the concentration levels in experiments designed to mimic pathological conditions.     

Nitrosative protein tyrosine modifications: biochemistry and functional significance

Nitrosative modifications regulate cellular signal transduction and pathogenesis of inflammatory responses and neurodegenerative diseases. Protein tyrosine nitration is a biomarker of oxidative stress and also influences protein structure and function. Recent advances in mass spectrometry have made it possible to identify modified proteins and specific modified amino acid residues. For analysis of nitrated peptides with low yields or only a subset of peptides, affinity 'tags' can be bait for 'fishing out' target analytes from complex mixtures. These tagged peptides are then extracted to a solid phase, followed by mass analysis. In this review, we focus on protein tyrosine modifications caused by nitrosative stresses and proteomic methods for selective enrichment and identification of nitrosative protein modifications.     

Proteomic analysis of 4-hydroxynonenal and nitrotyrosine modified proteins in RTT fibroblasts

Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births. A clear etiological factor present in more than 90% of classical RTT cases is the mutation of the gene encoding methyl-CpG-binding protein 2 (MECP2). Recent work from our group was able to shown a systemic oxidative stress (OxS) in these patients that correlates with the gravity of the clinical features.Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have performed a two-dimensional gel electrophoresis in order to evidence the oxidative modifications of proteins with special focus on the formation of protein adducts with 4-hydroxynonenal (4-HNE PAs)—a major secondary product of lipid peroxidation— and Nitrotyrosine, a marker derived from the biochemical interaction of nitric oxide (NO) or nitric oxide-derived secondary products with reactive oxygen species (ROS). Then, oxidatively modified spots were identified by mass spectrometry, LC-ESI-CID-MS/MS.Our results showed that 15 protein spots presented 4-HNE PAs and/or nitrotyrosine adducts in fibroblasts proteome from RTT patients compared to healthy control cells. Post-translationally modified proteins were related to several functional categories, in particular to cytoskeleton structure and protein folding. In addition, clear upregulated expression of the inducible NO synthase (iNOS) with high nitrite levels were observed in RTT fibroblasts, justifying the increased nitrotyrosine protein modifications.The present work describes not only the proteomic profile in RTT fibroblasts, but also identifies the modified proteins by 4-HNE and nitrotyrosine. Of note, for the first time, it appears that a dysregulation of NO pathway can be associated to RTT pathophysiology. In conclusion, the evidence of a wide range of proteins able to forms adducts with 4-HNE, Nitrotyrosine or with both confirms the possible alteration of several aspects of cellular functions that well correlates to the complex clinical features of RTT patients.     

Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides

Reaction of radicals in the presence of O2, and singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, and that this can result in inactivation of thiol-dependent enzymes. The major route for the cellular removal of damaged proteins is via catabolism mediated by proteosomal and lysosomal pathways; cysteine proteases (cathepsins) play a key role in the latter system. We hypothesized that inactivation of cysteine proteases by hydroperoxide-containing oxidised proteins may contribute to the accumulation of modified proteins within cells. We show here that thiol-dependent cathepsins, either isolated or in cell lysates, are rapidly and efficiently inactivated by amino acid, peptide, and protein hydroperoxides in a time- and concentration-dependent manner; this occurs with similar efficacy to equimolar H2O2. Inactivation involves reaction of the hydroperoxide with Cys residues as evidenced by thiol loss and formation of sulfenic acid intermediates. Structurally related, non-thiol-dependent cathepsins are less readily inactivated by these hydroperoxides. This inhibition, by oxidized proteins, of the system designed to remove modified proteins, may contribute to the accumulation of damaged proteins in cells subject to oxidative stress.     

Phenotypic variations in blood and milk of the Somali camel

132 blood samples and 54 milk samples obtained from Somali camel were analysed for red blood cell antigens with the cattle reagents and for Hb, Ca, X proteins, Tf, Alb, Am, SOD, alpha-La, beta-Lg and casein systems respectively. Positive lytic reactions were obtained with the anti-B, -Q, -Q', -W, -F1 and -J reagents. No biochemical polymorphism was observed except for Hb, X protein and beta-Lg systems.