RESUMO
Protein cross-links are formed in regulated biochemical processes in many biological systems, but they are also generated inadvertently via the reactions of exogenous or endogenous oxidants. Site-specific identification and characterization of such cross-links is challenging, and the goal was, therefore, to develop mass-spectrometry-based approaches tailored for proteins subjected to oxidative challenges that also are applicable for the analysis of complex samples. Using trypsin-mediated 18O isotopic labeling, different types of data acquisition workflows, and designated database software tools, we successfully identified tyrosine-tyrosine, tyrosine-tryptophan, tyrosine-lysine, and histidine-lysine cross-links in proteins subjected to sensitizer-mediated photo-oxidation with rose bengal or chemical oxidation with peroxyl radicals generated from the water-soluble compound 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Subsequently, AAPH was also applied to a protein extract from the Gram-positive bacterium Lactococcus lactis, demonstrating the feasibility to identify tyrosine-tyrosine, tyrosine-tryptophan, and tryptophan-tryptophan cross-linked peptides in a complex system. Different fragmentation techniques were evaluated, and it was observed that higher-energy collisional dissociation (HCD) resulted in a higher number of identified cross-link peptides, while electron-transfer dissociation supplemented with HCD (EThcD) generally provides higher fragment ion coverage of the cross-linked peptides.
Assuntos
Reagentes de Ligações Cruzadas , Peptídeos/análise , Proteínas/metabolismo , Técnica de Seleção de Aptâmeros , Espectrometria de Massas em Tandem/métodos , Aptâmeros de Peptídeos , Fluoresceínas , Marcação por Isótopo , Oxirredução , Peróxidos , Proteínas/análiseRESUMO
Metal ion-catalyzed oxidation of hydrazine and its derivatives leads to the formation of the hydrazyl radical and subsequently to oxy-radicals in the presence of molecular oxygen. Here, we have examined the role of Cu(2+)-catalyzed oxidation of hydralazine in the induction of DNA damage. Neither 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) nor dimethyl sulfoxide (DMSO) was effective in inhibiting hydralazine-Cu(2+)-induced DNA damage. Singlet oxygen did not appear to participate in this DNA cleavage. The one-electron oxidation of hydralazine also leads to the formation of DNA radicals as confirmed by immuno-spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide. Electron spin resonance (ESR) and spin-trapping studies further confirmed the formation of DNA radicals; predominantly, 2'-deoxyadenosine radical adducts were detected, while some radicals were also detected with other nucleosides. Our results suggest that free hydroxyl radicals may not be the main damaging species causing DNA cleavage and that possibly Cu-peroxide complexes, formed from Cu(+)-H2O2, are responsible for this hydralazine-Cu(2+)-induced DNA cleavage.
Assuntos
Cobre/química , Clivagem do DNA , DNA/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hidralazina/química , Marcadores de Spin , Dano ao DNA , Oxigênio/químicaRESUMO
The mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROOâ) and peroxynitrite (ONOO-), were explored. G6PDH was incubated with AAPH (2,2' -azobis(2-methylpropionamidine)dihydrochloride), used as ROOâ source, and ONOO-. Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaphthalene-8-sulfonic acid). Incubation of G6PDH (54.4 µM) with 60 mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to â¼59% of the initial level after 90 min. ONOO- induced a significant and concentration-dependent loss of G6PDH activity with â¼46% of the initial activity lost on treatment with 1.5 mM ONOO-. CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROOâ, but not ONOO-. LC-MS analysis provided evidence for ONOO--mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROOâ. These two oxidant systems therefore have markedly different effects on G6PDH structure and activity.
Assuntos
Aminoácidos , Leuconostoc mesenteroides , Aminoácidos/química , Glucosefosfato Desidrogenase/química , Oxidantes/química , Oxirredução , Peróxidos , Ácido Peroxinitroso , Desdobramento de ProteínaRESUMO
Nitrone spin traps such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are commonly used for free radical detection. Though proven examples are rare, artifact formation must be considered. For example, the Forrester-Hepburn mechanism yields the same radical adduct as that formed by genuine radical trapping. A hydroxylamine is formed by nucleophilic attack of the substrate on DMPO and subsequently oxidized to the respective nitroxide radical. One potential candidate for this artifact is the sulfur trioxide radical adduct (DMPO/(â¢)SO(3)(-)), as detected in spin-trapping experiments with horseradish peroxidase and sulfite. It has previously been shown by NMR experiments that the hydroxylamine intermediate does indeed form, but no direct proof for the ESR artifact has been provided. Here, we used isotopically labeled DMPO with horseradish peroxidase and ferricyanide to test for the Forrester-Hepburn artifact directly in a spin-trapping experiment. Besides sulfite, we investigated other nucleophiles such as cyanide, cysteine, and glutathione. Neither sulfite nor biological thiols produced detectable spin-trapping artifacts, but with cyanide the relatively weak signal originated entirely from the nucleophilic reaction. The hydroxylamine intermediate, which is more abundant with cyanide than with sulfite, was identified as cyano-hydroxylamine by means of 2D NMR experiments. Although our study found that spin trapping provided authentic free radical signals with most of the substrates, the occurrence of the Forrester-Hepburn mechanism artifact with cyanide emphasizes the importance of isotope measurements with nucleophile substrates.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Detecção de Spin , Óxidos N-Cíclicos/química , Cisteína/química , Ferricianetos/química , Glutationa/química , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/química , Hidroxilamina/síntese química , Hidroxilamina/química , Óxido Nítrico/química , Óxidos de Nitrogênio/química , Oxirredução , Sulfitos/químicaRESUMO
5,5-Dimethylpyrroline N-oxide (15N) and 5,5-di(trideuteromethyl)pyrroline N-oxide were synthesized from the respective isotopically labeled 2-nitropropane analogs obtained from the reaction of sodium nitrate with 2-halopropanes. This facile, straightforward process allows synthesizing isotopically labeled DMPO analogs in a 4-step reaction without special equipment.
Assuntos
Óxidos N-Cíclicos/síntese química , Nitroparafinas/química , Propano/análogos & derivados , Isótopos de Carbono/química , Óxidos N-Cíclicos/química , Marcação por Isótopo , Espectrometria de Massas , Nitratos/química , Isótopos de Nitrogênio/química , Propano/química , Marcadores de Spin , Detecção de Spin/métodosRESUMO
The present work examined the oxidation and crosslinking of the anti-bacterial enzyme lysozyme (Lyso), which is present in multiple biological fluids, and released from the cytoplasmic granules of macrophages and neutrophils at sites of infection and inflammation. It is therefore widely exposed to oxidants including peroxyl radicals (ROOâ¢). We hypothesized that exposure to ROO⢠would generate specific modifications and inter- and intra-protein crosslinks via radical-radical reactions. Lyso was incubated with AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) as a ROO⢠source. Enzymatic activity was assessed, while oxidative modifications were detected and quantified using electrophoresis and liquid chromatography (UPLC) with fluorescence or mass detection (MS). Computational models of AAPH-Lyso interactions were developed. Exposure of Lyso to AAPH (10 and 100 mM for 3 h, and 20 mM for 1 h), at 37 °C, decreased enzymatic activity. 20 mM AAPH showed the highest efficiency of Lyso inactivation (1.78 mol of Lyso inactivated per ROOâ¢). Conversion of Met to its sulfoxide, and to a lesser extent, Tyr oxidation to 3,4-dihydroxyphenylalanine and diTyr, were detected by UPLC-MS. Extensive transformation of Trp, involving short chain reactions, to kynurenine, oxindole, hydroxytryptophan, hydroperoxides or di-alcohols, and N-formyl-kynurenine was detected, with Trp62, Trp63 and Trp108 the most affected residues. Interactions of AAPH inside the negatively-charged catalytic pocket of Lyso, with Trp108, Asp52, and Glu35, suggest that Trp108 oxidation mediates, at least partly, Lyso inactivation. Crosslinks between Tyr20-Tyr23 (intra-molecular), and Trp62-Tyr23 (inter-molecular), were detected with both proximity (Tyr20-Tyr23), and chain flexibility (Trp62) appearing to favor the formation of covalent crosslinks.
Assuntos
Muramidase , Tirosina , Amidinas , Cromatografia Líquida , Radicais Livres , Muramidase/metabolismo , Oxirredução , Peróxidos , Espectrometria de Massas em TandemRESUMO
Photosensitized protein oxidation is a promising tool for medical procedures such as photochemical tissue bonding (PTB). We have recently reported that the binding of rose Bengal, a sensitizer employed in PTB, to lysozyme modulates the photooxidation and crosslinking of this protein. In this work we examined the photooxidation and crosslinking of lysozyme mediated by riboflavin (RF) an endogenous sensitizer also employed in PTB. We hypothesized that since RF does not bind strongly to proteins, the mechanism(s) and extent of enzymatic inactivation, amino acid modification and protein crosslinking would be dependent on the presence of O2, and differ to that induced by rose Bengal. This hypothesis was tested using UV-visible spectrophotometry, isothermal titration calorimetry (ITC), SDS-PAGE gels, quantification of amino acid consumption, and LC-MS analysis of sites of modification and crosslinks. Under N2, limited damage was detected arising from type 1 (radical) chemistry with formation of specific intra- (Tyr20-Tyr23) and inter- (Tyr23-Trp108) molecular crosslinks. In contrast, the presence of O2 triggered extensive protein damage through mixed type 1 and type 2 (1O2) mechanisms leading to Trp, Met, Tyr and His oxidation, loss of enzymatic activity and protein dimerization. LC-MS analysis provided evidence for crosslinking via radical-radical recombination reactions (Trp28-Tyr53), and secondary reactions involving nucleophilic attack of the side-chain amine of Lys116 on carbonyl groups. Overall, this behavior is in marked contrast to that detected with rose Bengal indicating that the mechanisms and sites of photo-oxidative damage, and consequences for protein function, can be modulated by the choice of sensitizing dye.
Assuntos
Muramidase , Riboflavina , Aminoácidos , Oxirredução , Rosa BengalaRESUMO
UV light is a widely-employed, and environmentally-sensitive bactericide but its mechanism of action is not fully defined. Proteins are major chromophores and targets for damage due to their abundance, but the role of proteins in inducing damage to bound DNA, and the effects on DNA-protein interactions is less well characterized. In E. coli (and other Gram-negative bacteria) the cyclic AMP receptor protein (CRP/CAP) regulates more than 500 genes. In this study we show that exposure of isolated dimeric CRP-cAMP to UV modifies specific Met, Trp, Tyr, and Pro side-chains, induces inter-protein Tyr63-Tyr41 cross-links, and decreases DNA binding via oxidation of Met114/Pro110 residues in close proximity at the CRP dimer interface. UV exposure also modifies DNA-bound cAMP-CRP, with this resulting in DNA cleavage at specific G/C residues within the sequence bound to CRP, but not at other G/C sites. Oxidation also increases CRP dissociation from DNA. The modifications at the CRP dimer interface, and the site-specific DNA strand cleavage are proposed to occur via oxidation of two species Met residues (Met114 and Met189, respectively) to reactive persulfoxides that damage neighbouring amino acids and DNA bases. These data suggest that modification to CRP, and bound DNA, contributes to UV sensitivity.
Assuntos
Proteína Receptora de AMP Cíclico/metabolismo , DNA Bacteriano/química , Escherichia coli/efeitos da radiação , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Raios Ultravioleta , Sítios de Ligação , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Dimerização , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Espectrometria de Massas , Oxigênio/química , Plasmídeos/genética , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , ProteômicaRESUMO
Modification of proteins in infant milk formula (IF) is of major concern to the dairy industry and consumers. Thermal treatment is required for microbiological safety, but heat, light, metal-ions and other factors may induce oxidative damage, and be a health risk. In this study protein modifications in IFs were quantified. IFs contained both reducible (disulphide) and non-reducible (di-tyrosine, lanthionine, lysinoalanine) protein cross-links. Dehydroalanine and the cross-linked species lanthionine and lysinoalanine were detected. Protein carbonyls were detected predominantly on high molecular mass materials. Oxidation products of phenylalanine (m-tyrosine), tryptophan (N-formylkynurenine, kynurenine, 3-hydroxykynurenine), tyrosine (di-tyrosine) and methionine (methionine sulphoxide) were detected, consistent with amino acid modification. Higher levels of most of the markers of protein modification were present in the hydrolysed protein brand, when compared to the conventional IF samples, indicative of increased damage during additional processing. Significant levels of racemised (D-) amino acids were present. These data indicate that amino acids in proteins in IFs are modified to a significant extent during manufacture, with hydrolysed IF being particularly prone.
Assuntos
Aminoácidos/metabolismo , Fórmulas Infantis/química , Proteínas do Leite/análise , Aminoácidos/análise , Animais , Humanos , Hidrólise , Lactente , Proteínas do Leite/metabolismo , Oxirredução , PósRESUMO
This work examined the hypothesis that interactions of Rose Bengal (RB2-) with lysozyme (Lyso) might mediate type 1 photoreactions resulting in protein cross-linking even under conditions favoring 1O2 formation. UV-visible spectrophotometry, isothermal titration calorimetry (ITC), and docking analysis were employed to characterize RB2--Lyso interactions, while oxidation of Lyso was studied by SDS-PAGE gels, extent of amino acid consumption, and liquid chromatography (LC) with mass detection (employing tryptic peptides digested in H218O and H2O). Docking studies showed five interaction sites including the active site. Hydrophobic interactions induced a red shift of the visible spectrum of RB2- giving a Kd of 4.8⯵M, while data from ITC studies, yielded a Kd of 0.68⯵M as an average of the interactions with stoichiometry of 3.3 RB2- per Lyso. LC analysis showed a high consumption of readily-oxidized amino acids (His, Trp, Met and Tyr) located at different and diverse locations within the protein. This appears to reflect extensive damage on the protein probably mediated by a type 2 (1O2) mechanism. In contrast, docking and mass spectrometry analysis provided evidence for the generation of specific intra- (Tyr23-Tyr20) and inter-molecular (Tyr23-Trp62) Lyso cross-links, and Lyso dimer formation via radical-radical, type 1 mechanisms.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Corantes Fluorescentes/metabolismo , Muramidase/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Rosa Bengala/metabolismo , Triptofano/química , Tirosina/química , Animais , Galinhas , Reagentes de Ligações Cruzadas/química , Corantes Fluorescentes/química , Muramidase/química , Oxirredução , Fotoquímica , Fármacos Fotossensibilizantes/química , Conformação Proteica , Rosa Bengala/químicaRESUMO
Oxidation can be induced by multiple processes in biological samples, with proteins being important targets due to their high abundance and reactivity. Oxidant reactions with proteins are not comprehensively understood, but it is known that structural and functional changes may be a cause, or a consequence, of disease. The mechanisms of oxidation of the model protein RNAse A by singlet oxygen (1O2) were examined and compared to peroxyl radical (ROOâ¢) oxidation, both common biological oxidants. This protein is a prototypic member of the RNAse family that exhibits antiviral activity by cleaving single-stranded RNA. RNAse A lacks tryptophan and cysteine residues which are major oxidant targets, but contains multiple histidine, tyrosine and methionine residues; these were therefore hypothesized to be the major sites of damage. 1O2 and ROO⢠induce different patterns and extents of damage; both induce cross-links and side-chain oxidation, and 1O2 exposure modulates enzymatic activity. Multiple products have been characterized including methionine sulfoxide and sulfone, alcohols, DOPA, 2-oxohistidine, histidine-derived ring-opened species and inter- and intra-molecular cross-links (di-tyrosine, histidine-lysine, histidine-arginine, tyrosine-lysine). In addition to methionine modification, which appears not to be causative to activity loss, singlet oxygen also induces alteration to specific histidine, tyrosine and proline residues, including modification and cross-linking of the active site histidine, His12. The high homology among the RNAse family suggests that similar modifications may occur in humans, and be associated with the increased risk of viral infections in people with diabetes, as markers for 1O2 have been found in early stages of this pathology.
Assuntos
Peróxidos/metabolismo , Ribonuclease Pancreático/química , Oxigênio Singlete/metabolismo , Relação Estrutura-Atividade , Histidina/química , Humanos , Oxidantes/química , Oxirredução , Conformação Proteica , Ribonuclease Pancreático/metabolismo , Tirosina/químicaRESUMO
Previous studies on metal-ion catalyzed oxidation of α-synuclein oxidation have mostly used conditions that result in extensive modification precluding an understanding of the early events in this process. In this study, we have examined time-dependent oxidative events related to α-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 4.7:15.6:1 gave 0.22 carbonyls per α-synuclein within 15â¯min. With the latter conditions, rapid conversion of 3 out of 4 methionines (Met) to methionine sulfoxide, and 2 out of 4 tyrosines (Tyr) were converted to products including inter- and intra-molecular dityrosine cross-links and protein oligomers, as determined by SDS-PAGE and Western blot analysis. Limited histidine (His) modification was observed. The rapid formation of dityrosine cross-links was confirmed by fluorescence and mass-spectrometry. These data indicate that Met and Tyr oxidation are early events in Cu2+/H2O2-mediated damage, with carbonyl formation being a minor process. With the Cu2+/H2O2/ascorbate system, rapid protein carbonyl formation was detected with the first 5â¯min, but after this time point, little additional carbonyl formation was detected. With this system, lower levels of Met and Tyr oxidation were detected (2 Met and 1 Tyr modified with a Cu2+/H2O2/ascorbate/protein ratio of 2.3:7.8:7.8:1), but greater His oxidation. Only low levels of intra- dityrosine cross-links and no inter- dityrosine oligomers were detected under these conditions, suggesting that ascorbate limits Cu2+/H2O2-induced α-synuclein modification.
Assuntos
Cobre/farmacologia , Metionina/análogos & derivados , Tirosina/análogos & derivados , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Catálise , Humanos , Peróxido de Hidrogênio/farmacologia , Metionina/química , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Oligoelementos/farmacologia , Tirosina/químicaRESUMO
The present work examined the role of Tyr and Trp in oxidative modifications of caseins, the most abundant milk proteins, induced by peroxyl radicals (ROOâ¢). We hypothesized that the selectivity of ROO⢠and the high flexibility of caseins (implying a high exposure of Tyr and Trp residues) would favor radical-radical reactions, and di-tyrosine (di-Tyr) and di-tryptophan (di-Trp) formation. Solutions of α- and ß-caseins were exposed to ROO⢠from thermolysis and photolysis of AAPH (2,2'-azobis(2-methylpropionamidine)dihydrochloride). Oxidative modifications were examined using electrophoresis, western blotting, fluorescence, and chromatographic methodologies with diode array, fluorescence and mass detection. Exposure of caseins to AAPH at 37⯰C gave fragmentation, cross-linking and protein aggregation. Amino acid analysis showed consumption of Trp, Tyr, Met, His and Lys residues. Quantification of Trp and Tyr products, showed low levels of di-Tyr and di-Trp, together with an accumulation of carbonyls indicating that casein aggregation is, at least partly, associated with secondary reactions between carbonyls and Lys and His residues. AAPH photolysis, which generates a high flux of free radicals increased the extent of formation of di-Tyr in both model peptides and α- and ß- caseins; di-Trp was only detected in peptides and α-casein. Thus, in spite of the high flexibility of caseins, which would be expected to favor radical-radical reactions, the low flux of ROO⢠generated during AAPH thermolysis disfavours the formation of dimeric radical-radical cross-links such as di-Tyr and di-Trp, instead favoring other O2-dependent crosslinking pathways such as those involving secondary reactions of initial carbonyl products.
Assuntos
Amidinas/química , Caseínas/química , Fragmentos de Peptídeos/química , Peróxidos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Triptofano/química , Tirosina/química , Animais , Caseínas/classificação , Bovinos , Cinética , Oxidantes/química , Oxirredução , Peróxidos/químicaRESUMO
Protein oxidation is a frequent event as a result of the high abundance of proteins in biological samples and the multiple processes that generate oxidants. The reactions that occur are complex and poorly understood, but can generate major structural and functional changes on proteins. Current data indicate that pathophysiological processes and multiple human diseases are associated with the accumulation of damaged proteins. In this study we investigated the mechanisms and consequences of exposure of the key metabolic enzyme glucose-6-phosphate dehydrogenase (G6PDH) to peroxyl radicals (ROOâ¢) and singlet oxygen (1O2), with particular emphasis on the role of Trp and Tyr residues in protein cross-linking and fragmentation. Cross-links and high molecular mass aggregates were detected by SDS-PAGE and Western blotting using specific antibodies. Amino acid analysis has provided evidence for Trp and Tyr consumption and formation of oxygenated products (diols, peroxides, N-formylkynurenine, kynurenine) from Trp, and di-tyrosine (from Tyr). Mass spectrometric data obtained after trypsin-digestion in the presence of H216O and H218O, has allowed the mapping of specific cross-linked residues and their locations. These data indicate that specific Tyr-Trp and di-Tyr cross-links are formed from residues that are proximal and surface-accessible, and that the extent of Trp oxidation varies markedly between sites. Limited modification at other residues is also detected. These data indicate that Trp and Tyr residues are readily modified by ROO⢠and 1O2 with this giving products that impact significantly on protein structure and function. The formation of such cross-links may help rationalize the accumulation of damaged proteins in vivo.
Assuntos
Proteínas de Bactérias/química , Glucosefosfato Desidrogenase/química , Peróxidos/química , Oxigênio Singlete/química , Triptofano/química , Tirosina/química , Reagentes de Ligações Cruzadas/química , Ensaios Enzimáticos , Cinética , Cinurenina/análogos & derivados , Cinurenina/química , Leuconostoc mesenteroides/química , Leuconostoc mesenteroides/enzimologia , Luz , Oxirredução , Processos Fotoquímicos , Agregados Proteicos , SoluçõesRESUMO
FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROOâ¢) generated from AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROOâ¢. Quantification of the number of moles of amino acid consumed per mole of ROO⢠shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROOâ¢-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples.
Assuntos
Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Escherichia coli/metabolismo , Peróxidos/química , Triptofano/química , Tirosina/química , Amidinas/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reagentes de Ligações Cruzadas/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Expressão Gênica , Mutação , Oxidantes/química , Oxirredução , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triptofano/metabolismo , Tirosina/metabolismoRESUMO
Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H2O2) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H2O2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O2â¢-) and H2O2 in the presence of NADH. Generation of the free radical O2â¢- and H2O2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/metabolismo , Superóxidos/metabolismo , Catálise , Células HEK293 , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Luminescentes/metabolismo , NAD/metabolismo , Estresse Oxidativo , Proteína Vermelha FluorescenteRESUMO
Parkinson's disease (PD) is a debilitating, progressive, neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and motor deficits. Alpha-synuclein-containing aggregates represent a feature of a variety of neurodegenerative disorders, including PD; however, the mechanism that initiates and promotes intraneuronal alpha-synuclein aggregation remains unknown. We hypothesized protein radical formation as an initiating mechanism for alpha-synuclein aggregation. Therefore, we used the highly sensitive immuno-spin trapping technique to investigate protein radical formation as a possible mechanism of alpha-synuclein aggregation as well as to investigate the source of protein radical formation in the midbrains of Maneb- and paraquat-coexposed mice. Coexposure to Maneb and paraquat for 6 weeks resulted in active microgliosis, NADPH oxidase activation, and inducible nitric oxide synthase (iNOS) induction, which culminated in protein radical formation in the midbrains of mice. Results obtained with immuno-spin trapping and immunoprecipitation experiments confirmed formation of alpha-synuclein radicals in dopaminergic neurons of exposed mice. Free radical formation requires NADPH oxidase and iNOS, as indicated by decreased protein radical formation in knockout mice (P47phox(-/-) and iNOS(-/-)) and in mice treated with inhibitors such as FeTPPS (a peroxynitrite decomposition catalyst), 1400 W (an iNOS inhibitor), or apocynin (a NADPH oxidase inhibitor). Concurrence of protein radical formation with dopaminergic neuronal death indicated a link between protein radicals and disease progression. Taken together, these results show for the first time the formation and detection of the alpha-synuclein radical and suggest that NADPH oxidase and iNOS play roles in peroxynitrite-mediated protein radical formation and subsequent neuronal death in the midbrains of Maneb- and paraquat-coexposed mice.
Assuntos
Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismo , Animais , Óxidos N-Cíclicos/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Injeções Intraperitoneais , Masculino , Maneb , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Modelos Biológicos , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Paraquat , Ácido Peroxinitroso/metabolismo , Marcadores de Spin , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Free radical detection with ESR spin trapping relies on the specific addition of the radical to nitrone/nitroso compounds. It also has been proposed that spin traps can react in biological systems to give false-positive results. For nitrone spin traps, the reaction with nucleophiles, first described by Forrester and Hepburn, has been discussed as the most critical source of artifacts. For artifact identification, the ESR preincubation method may be used, which employs isotopically marked spin traps. Here we investigated the influence of fast sulfite-hydroxylamine equilibrium chemistry on the validity of this assay. Using the (faster) aspiration technique, we found that the Forrester-Hepburn mechanism also contributes to DMPO/(â¢)SO3(-) adduct formation during ferricyanide-mediated sulfite oxidation, but no evidence for artifactual DMPO/(â¢)SO3(-) formation was found if the more potent horseradish peroxidase was used. This is ESR evidence that the Forrester-Hepburn mechanism can occur under mild conditions, depending on the experimental details. This technique can also be used to test for other artifact mechanisms. We investigated the known ene reaction of DBNBS and tryptophan in more detail. We found that a strong artifact signal is induced by light; however, with atypically long incubations, we found that the artifact is also formed thermally.
Assuntos
Artefatos , Benzenossulfonatos/química , Radicais Livres/análise , Compostos Nitrosos/química , Detecção de Spin/métodos , Triptofano/química , Benzenossulfonatos/análise , Óxidos N-Cíclicos/química , Espectroscopia de Ressonância de Spin Eletrônica , Reações Falso-Positivas , Ferricianetos/química , Peroxidase do Rábano Silvestre/química , Hidroxilamina/química , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Óxidos de Nitrogênio/química , Compostos Nitrosos/análise , Oxirredução , Marcadores de Spin , Sulfitos/químicaRESUMO
Although it is widely known that arsenic-contaminated drinking water causes many diseases, arsenic's exact mode of action (MOA) is not fully understood. Induction of oxidative stress has been proposed as an important key event in the toxic MOA of arsenic. The authors' studies are centered on identifying a reactive species involved in the genotoxicity of arsenic using a catalase (CAT) knockout mouse model that is impaired in its ability to breakdown hydrogen peroxide (H2 O2 ). The authors assessed the induction of DNA damage using the Comet assay following exposure of mouse Cat(+/) (+) and Cat(-) (/) (-) primary splenic lymphocytes to monomethylarsonous acid (MMA(III) ) to identify the potential role of H2 O2 in mediating cellular effects of this metalloid. The results showed that the Cat(-) (/) (-) lymphocytes are more susceptible to MMA(III) than the Cat(+/) (+) lymphocytes by a small (1.5-fold) but statistically significant difference. CAT activity assays demonstrated that liver tissue has approximately three times more CAT activity than lymphocytes. Therefore, Comet assays were performed on primary Cat(+/) (+) , Cat(+/) (-) , and Cat(-) (/) (-) hepatocytes to determine if the Cat(-) (/) (-) cells were more susceptible to MMA(III) than lymphocytes. The results showed that the Cat(-) (/) (-) hepatocytes exhibit higher levels of DNA strand breakage than the Cat(+/) (+) (approximately fivefold) and Cat(+/) (-) (approximately twofold) hepatocytes exposed to MMA(III) . Electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as the spin-trap agent detected the generation of ·OH via MMA(III) when H2 O2 was present. These experiments suggest that CAT is involved in protecting cells against the genotoxic effects of the ·OH generated by MMA(III) .