RESUMO
Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.
Assuntos
Glutamato-Amônia Ligase , Ácido Peroxinitroso , Processamento de Proteína Pós-Traducional , Humanos , Cromatografia Líquida , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/farmacologia , Espectrometria de Massas em Tandem , Tirosina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Oxirredução , Mutação , Agregação Patológica de Proteínas/induzido quimicamenteRESUMO
Fibronectin (FN) is an abundant glycoprotein found in plasma and the extracellular matrix (ECM). It is present at high concentrations at sites of tissue damage, where it is exposed to oxidants generated by activated leukocytes, including peroxynitrous acid (ONOOH) formed from nitric oxide (from inducible nitric oxide synthase) and superoxide radicals (from NADPH oxidases and other sources). ONOOH reacts rapidly with the abundant tyrosine and tryptophan residues in ECM proteins, resulting in the formation of 3-nitrotyrosine, di-tyrosine, and 6-nitrotryptophan. We have shown previously that human plasma FN is readily modified by ONOOH, but the extent and location of modifications, and the role of FN structure (compact versus extended) in determining these factors is poorly understood. Here, we provide a detailed LC-MS analysis of ONOOH-induced FN modifications, including the extent of their formation and the sites of intramolecular and intermolecular cross-links, including Tyr-Tyr, Trp-Trp, and Tyr-Trp linkages. The localization of these cross-links to specific domains provides novel data on the interactions between different modules in the compact conformation of plasma FN and allows us to propose a model of its unknown quaternary structure. Interestingly, the pattern of modifications is significantly different to that generated by another inflammatory oxidant, HOCl, in both extent and sites. The characterization and quantification of these modifications offers the possibility of the use of these materials as specific biomarkers of ECM modification and turnover in the many pathologies associated with inflammation-associated fibrosis.
Assuntos
Fibronectinas/metabolismo , Fibronectinas/fisiologia , Ácido Peroxinitroso/química , Aterosclerose/metabolismo , Células Cultivadas , Cromatografia em Gel/métodos , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Fibronectinas/química , Humanos , Inflamação/metabolismo , Oxidantes/metabolismo , Oxirredução , Ácido Peroxinitroso/farmacologia , Domínios Proteicos/fisiologia , Triptofano/análogos & derivados , Triptofano/química , Tirosina/análogos & derivados , Tirosina/químicaRESUMO
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
INTRODUCTION: Protein cross-links are common in biological systems and are generated both deliberately as a part of normal metabolism and also accidently as a result of exposure to external factors such as oxidation and glycation stresses. These cross-links can be both positive and negative for biological function, with high levels of inappropriate cross-links being associated with multiple human pathologies, as well as loss of protein structure and function in the food, agricultural, and pharmaceutical fields. Areas covered: This review covers recent advances in the detection and identification of protein cross-links arising from oxidation reactions, mediated by both radicals and two-electron oxidants. Information on both enzymatic and nonenzymatic cross-linking is reviewed, both where this is intentional, as part of normal metabolism, and accidental and a potential cause of disease. Expert commentary: The advantages and drawbacks of various methods available for the detection, identification, and quantification of these species are discussed, as well as some of the mechanisms and processes known to give rise to these species.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Proteínas/metabolismo , Algoritmos , Humanos , Espectrometria de Massas , Oxidantes/metabolismo , OxirreduçãoRESUMO
Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen (1O2)-mediated oxidation of multiple proteins (α-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDS-PAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the 1O2 concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with 18O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta-2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide 'proof-of-concept' for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies.
Assuntos
Dissulfetos , Oxigênio Singlete , Glutationa/metabolismo , Humanos , Oxirredução , ProteínasRESUMO
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
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
Elastin is an abundant extracellular matrix protein in elastic tissues, including the lungs, skin and arteries, and comprises 30-57% of the aorta by dry mass. The monomeric precursor, tropoelastin (TE), undergoes complex processing during elastogenesis to form mature elastic fibres. Peroxynitrous acid (ONOOH), a potent oxidising and nitrating agent, is formed in vivo from superoxide and nitric oxide radicals. Considerable evidence supports ONOOH formation in the inflamed artery wall, and a role for this species in the development of human atherosclerotic lesions, with ONOOH-damaged extracellular matrix implicated in lesion rupture. We demonstrate that TE is highly sensitive to ONOOH, with this resulting in extensive dimerization, fragmentation and nitration of Tyr residues to give 3-nitrotyrosine (3-nitroTyr). This occurs with equimolar or greater levels of oxidant and increases in a dose-dependent manner. Quantification of Tyr loss and 3-nitroTyr formation indicates extensive Tyr modification with up to two modified Tyr per protein molecule, and up to 8% conversion of initial ONOOH to 3-nitroTyr. These effects were modulated by bicarbonate, an alternative target for ONOOH. Inter- and intra-protein di-tyrosine cross-links have been characterized by mass spectrometry. Examination of human atherosclerotic lesions shows colocalization of 3-nitroTyr with elastin epitopes, consistent with TE or elastin modification in vivo, and also an association of 3-nitroTyr containing proteins and elastin with lipid deposits. These data suggest that exposure of TE to ONOOH gives marked chemical and structural changes to TE and altered matrix assembly, and that such damage accumulates in human arterial tissue during the development of atherosclerosis.
Assuntos
Artérias/fisiologia , Aterosclerose/metabolismo , Inflamação/metabolismo , Macrófagos/fisiologia , Ácido Peroxinitroso/metabolismo , Tropoelastina/metabolismo , Tirosina/metabolismo , Aterosclerose/patologia , Cadáver , Elastina/metabolismo , Matriz Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Inflamação/patologia , Nitrocompostos/metabolismo , Oxirredução , Conformação Proteica , Tirosina/análogos & derivados , Resposta a Proteínas não DobradasRESUMO
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.