Fabian Leinisch

Peroxyl radical- and photo-oxidation of glucose 6- phosphate dehydrogenase generates cross-links and functional changes via oxidation of tyrosine and tryptophan residues

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 (ROO•) 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.

α- and β- casein aggregation induced by riboflavin-sensitized photo-oxidation occurs via di-tyrosine cross-links and is oxygen concentration dependent

Type I photo-oxidation generates Trp-(TrpN) and Tyr-derived (TyrO) radicals in proteins which can dimerize producing cross-links, or alternatively react with O2. It was therefore hypothesized that the O2 concentration may have a significant effect on dye-photosensitized reactions. We studied photo-oxidation of α- and β-caseins induced by riboflavin (RF), a photosensitizing vitamin present in milk, under aerobic and anaerobic conditions. Triplet-state RF induced oxidative modifications on both caseins, and significant levels of cross-links. The extent of damage, and the yield of cross-links versus oxidized products, was dependent on the O2 concentration. In the absence of O2, the overall extent of damage was decreased, but the yield of cross-linked products was significantly elevated. These cross-links are consistent with inter- and intra-molecular di-Tyr or di-Trp bridges. Alternative cross-links were detected in the presence of O2, consistent with pathways involving the reaction of protein radicals with O2 or O2-.

Mass-Spectrometry-Based Identification of Cross-Links in Proteins Exposed to Photo-Oxidation and Peroxyl Radicals Using 18O Labeling and Optimized Tandem Mass Spectrometry Fragmentation

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.

Structural and functional changes in RNAse A originating from tyrosine and histidine cross-linking and oxidation induced by singlet oxygen and peroxyl radicals

Abstract 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 (ROO•) 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. Graphical abstract Figure. No caption available. HighlightsRNAse A was oxidized by singlet oxygen (1O2) and peroxyl radicals (ROO•).1O2, but not ROO•, decrease enzymatic activity.Oxidative modifications have been quantified and mapped to protein sequence.Met residues are major targets for oxidation, but appear not to be causative for activity loss.Multiple His‐ and Tyr‐derived cross‐links are formed, including active site His‐Arg links.

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

ABSTRACT Previous studies on metal–ion catalyzed oxidation of &agr;–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 &agr;–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 &agr;–synuclein within 15min. 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 5min, 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 &agr;–synuclein modification. HIGHLIGHTSMet and Tyr oxidation are early and rapid events in Cu2+/H2O2– mediated damage to &agr;–synuclein.Met sulfoxide and dityrosine detected within 5min of Cu2+/H2O2 exposure to &agr;–synuclein.Carbonyl formation is a minor process in early Cu2+/H2O2– mediated damage to &agr;–synuclein.Ascorbate limits Met and Tyr oxidation in Cu2+/H2O2/AscH‐–mediated damage to &agr;–synuclein.

The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function

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 (ROO•) 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 ROO•. 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 ROO•-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.

Aggregation of α- and β- caseins induced by peroxyl radicals involves secondary reactions of carbonyl compounds as well as di-tyrosine and di-tryptophan formation

Abstract The present work examined the role of Tyr and Trp in oxidative modifications of caseins, the most abundant milk proteins, induced by peroxyl radicals (ROO•). 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 &agr;‐ and &bgr;‐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 &agr;‐ and &bgr;‐ caseins; di‐Trp was only detected in peptides and &agr;‐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. Graphical abstract Figure. No Caption available. Highlights&agr;‐ and &bgr;‐caseins are extensively oxidized by peroxyl radicals.Protein crosslinking and aggregation are the most significant pathways.Extensive oxidation of Tyr and Trp was evidenced.Low fluxes of peroxyl radicals favor Schiff bases rather than di‐Tyr and di‐Trp.