Oxidation of whey protein isolate after thermal convection and microwave heating and freeze-drying: Correlation among physicochemical and NIR spectroscopy analyses.

This study investigated the oxidative susceptibility of whey protein isolate (WPI) dispersions treated by microwave or thermal convection before freeze-drying. WPI (20 mg protein/mL) in distilled water (DW) was heated at 63 ± 2 °C for 30 min by microwave (WPI-MW) or convection heating (WPI-CH) and freeze-dried. Untreated WPI (WPI-C), WPI solubilized in DW and freeze-dried (WPI-FD), and WPI solubilized in DW, heated at 98 ± 2 °C for 2 min and freeze-dried (WPI-B) were also evaluated. Structural changes (turbidity, ζ potential, SDS-PAGE, and near-infrared spectroscopy (NIR)) and protein oxidation (dityrosine, protein carbonylation, and SH groups) were investigated. WPI-FD showed alterations compared to WPI-C, mainly concerning carbonyl groups. Microwave heating increased carbonyl groups and dityrosine formation compared to conventional heating. NIR spectrum indicated changes related to the formation of carbonyl groups and PCA analysis allowed us to distinguish the samples according to carbonyl group content. The results suggest that NIR may contribute to monitoring oxidative changes in proteins resulting from processing.

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation.

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.

Free radicals derived from γ-radiolysis of water and AAPH thermolysis mediate oxidative crosslinking of eGFP involving Tyr-Tyr and Tyr-Cys bonds: the fluorescence of the protein is conserved only towards peroxyl radicals.

The enhanced green fluorescent protein (eGFP) is one of the most employed variants of fluorescent proteins. Nonetheless little is known about the oxidative modifications that this protein can undergo in the cellular milieu. The present work explored the consequences of the exposure of eGFP to free radicals derived from γ-radiolysis of water, and AAPH thermolysis. Results demonstrated that protein crosslinking was the major pathway of modification of eGFP towards these oxidants. As evidenced by HPLC-FLD and UPLC-MS, eGFP crosslinking would occur as consequence of a mixture of pathways including the recombination of two protein radicals, as well as secondary reactions between nucleophilic residues (e.g. lysine, Lys) with protein carbonyls. The first mechanism was supported by detection of dityrosine and cysteine-tyrosine bonds, whilst evidence of formation of protein carbonyls, along with Lys consumption, would suggest the formation and participation of Schiff bases in the crosslinking process. Despite of the degree of oxidative modifications elicited by peroxyl radicals (ROO•) generated from the thermolysis of AAPH, and free radicals generated from γ-radiolysis of water, that were evidenced at amino acidic level, only the highest dose of γ-irradiation (10 kGy) triggered significant changes in the secondary structure of eGFP. These results were accompanied by the complete loss of fluorescence arising from the chromophore unit of eGFP in γ-irradiation-treated samples, whereas it was conserved in ROO•-treated samples. These data have potential biological significance, as this fluorescent protein is widely employed to study interactions between cytosolic proteins; consequently, the formation of fluorescent eGFP dimers could act as artifacts in such experiments.

Exploring the role of lipids in protein modification and amyotrophic lateral sclerosis / Explorando o papel dos lipídeos na modificação de proteínas e esclerose lateral amiotrófica

Lipids are a diverse and ubiquitous group of compounds, which have several biological functions such as structural components of cell membranes, energy storage, and participation in signaling pathways. Free radicals or reactive oxygen species could attack polyunsaturated fatty acid esterified to phospholipids generating oxidized products. Once oxidized, lipids are able to modify amino acids residues in proteins leading to modulation signaling pathways and cellular redox balance. Furthermore, alteration of lipid homeostasis is also linked to development and progression of neurodegenerative diseases. The purposes of this study were (i) to investigate the role of lipids in protein aggregation, (ii) to investigate the plasma lipidome of an ALS rat model (SOD1G93A rats), and (iii) to investigate the effect of high-fat diet in plasma lipidome of an ALS rat model. In chapters 1 and 2, the interaction between cytochrome c (cytc) and cardiolipin hydroperoxide (CLOOH), as well as cholesterol hydroperoxide (ChOOH) promoted protein aggregation. Mass spectrometry analysis of tryptic peptides from CLOOH-containing reaction revealed K72 and H26 consistently modified by 4- hydroxynonenal (4-HNE). Further, adduction of K27, K73 and K88 were detected with 4- oxynonenal (4-ONE). For the first time, we characterized the dityrosine cross-linked peptides at Y48-Y74, Y48-97 and Y74-Y97 in oligomeric cytc. Similarly, ChOOH-containing reaction showed dityrosine cross-linked peptides at Y48-Y48, Y48-Y74 and Y48-Y97 in dimeric cytc. In accordance to previous studies, the proposed mechanism under covalent protein oligomerization mediated by lipid hydroperoxide could be related to modification of lysine and tyrosine residues. In chapter 3, we characterized the lipid composition of blood plasma in amyotrophic lateral sclerosis (ALS), since dysregulation of lipid metabolism is increasingly associated with neuropathology. Using untargeted lipidomics approach based on liquid chromatography coupled to mass spectrometry, we found main alterations in triglycerides, phospholipids and sphingolipids in symptomatic ALS rats relative to controls. Additionally, for the first time we reported acylceramides species in the plasma. In order to investigate the source of these lipid alterations, we analyzed the lipid content of fractioned lipoproteins. Triglycerides and phospholipids were found in very low-density lipoprotein (VLDL), while acylceramides and hexosylceramides were found enriched in high-density lipoprotein (HDL). In chapter 4, high-fat diet containing lard or high-fish oil as much as 60% of total lipids has both the largest change on plasma lipid composition. Overall survival was not statistically different when compared to control diet. Increased levels of acylceramides, hexosylceramides and acylcarnitines were observed in ALS rats fed a control diet or high-fat diet in comparison to WT controls. Importantly, untargeted lipidomic analysis of blood plasma highlighted acylceramide d18:1/24:1+20:4 as potential biomarkers of ALS progression. Thus, our lipidomic analysis provides a novel insight into the molecular level event driving molecular dysregulation in ALS. Additional research is needed to determine the effect of plasma lipid alteration on motor neuron process and energetic metabolism. Collectively, our findings reinforce the idea that lipids play a relevant role in modulating cellular processes linked to protein aggregation and neurodegeneration

Os lipídeos são moléculas que possuem várias funções biológicas importantes, atuando como componente de membranas celulares, servindo com fonte de reserva de energia e participando de vias de sinalização. Os ácidos graxos poli-insaturados esterificados aos fosfolipídeos, por exemplo, são potenciais alvos para o ataque de radicais livres gerando produtos oxidados que são capazes de modificar resíduos de aminoácidos em proteínas levando a modulação das vias de sinalização e balanço redox. Por outro lado, alteração na homeostase do metabolismo dos lipídeos está relacionada ao desenvolvimento e progressão de doenças neurodegenerativas. Tendo em vista a importância dos lipídeos nos processos biológicos, os objetivos desse estudo foram (i) investigar o papel dos lipídeos na agregação proteica (capítulo 1 e 2), (ii) investigar as alterações na composição lipídica do plasma de rato modelo SOD1G93A de esclerose lateral amiotrófica (ELA) (capítulo 3) e (iii) investigar o efeito da suplementação de dietas hiperlipídicas na composição lipídica do plasma de rato modelo SOD1G93A (capítulo 4). No capítulo 1 e 2, a interação do citocromo c (citc) com hidroperóxido de cardiolipina (CLOOH) e hidroperóxido de colesterol (ChOOH) promove a agregação covalente do citc. Análise por nLC-MS/MS dos peptídeos digeridos identificou resíduos de lisina (K72) e histidina (H26) modificado por 4-hidroxininenal (4-HNE), enquanto os resíduos K27, K73 e K88 foram modificados por 4-oxinonenal (4-ONE). Pela primeira vez, nós caracterizamos ditirosinas (Y48-Y74, Y48-97 e Y74-Y97) na reação do citc com CLOOH. Também foram caracterizadas ditirosinas envolvendo os resíduos Y48-Y48, Y48-Y74 e Y48-Y97 na reação com ChOOH. Esses resultados corroboram com estudos anteriores que sugerem um mecanismo de agregação proteica envolvendo a perda da carga positiva de lisina e formação de ditirosina pela combinação de radicais de tirosil. No capítulo 3, a análise da composição lipídica do plasma de ratos SOD1G93A utilizando LC-MS/MS revelou alterações significativas na composição de triglicérides, glicerofosfolipídeos e esfingolipídeos em ratos sintomáticos comparado com os assintomáticos. É importante destacar que pela primeira vez acilceramidas foram identificadas em plasma de rato modelo para ALS. Análise da composição lipídica de lipoproteínas isoladas, maior fonte de lipídeos circulantes no plasma, mostraram alterações de triglicérides e glicerofosfolipídeos em VLDL. As acilceramidas e as hexosilceramidas, por sua vez, foram encontradas em maior abundância em HDL. No capítulo 4, a suplementação com dietas hiperlipídicas (rica em banha de porco e óleo de peixe) alterou significativamente o perfil lipídico do plasma em relação a doença. Contudo, não foi observado aumento significativo na sobrevida dos ratos ALS comparado com dieta controle. Independente da dieta, a concentração plasmática de acilcarnitina, hexosilceramidas e acilceramidas foram significativamente aumentadas em ratos ALS comparado com WT. A análise do perfil lipídico do plasma mostrou que a acilceramida d18:1/24:1+20:4 pode ser um potencial marcador de progressão da ALS. Dessa forma, os resultados mostrados fornecem uma visão enriquecedora sobre o evento a nível molecular que conduz a desregulação lipídica na ELA. Coletivamente, nossos resultados reforçam a importância dos lipídeos na modulação dos processos celulares ligados a agregação de proteínas e na neurodegeneração

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

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 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.

α- and ß-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 ß-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-.

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.

Oxidation, inactivation and aggregation of protein disulfide isomerase promoted by the bicarbonate-dependent peroxidase activity of human superoxide dismutase.

Protein disulfide isomerase (PDI) is a dithiol-disulfide oxidoreductase that has essential roles in redox protein folding. PDI has been associated with protective roles against protein aggregation, a hallmark of neurodegenerative diseases. Intriguingly, PDI has been detected in the protein inclusions found in the central nervous system of patients of neurodegenerative diseases. Oxidized proteins are also consistently detected in such patients, but the agents that promote these oxidations remain undefined. A potential trigger of protein oxidation is the bicarbonate-dependent peroxidase activity of the human enzyme superoxide dismutase 1 (hSOD1). Therefore, we examined the effects of this activity on PDI structure and activity. The results showed that PDI was oxidized to radicals that lead to PDI inactivation and aggregation. The aggregates are huge and apparently produced by covalent cross-links. Spin trapping experiments coupled with MS analysis indicated that at least 3 residues of PDI are oxidized to tyrosyl radicals (Y(63), Y(116) and Y(327)). Parallel experiments showed that PDI is also oxidized to radicals, inactivated and aggregated by the action of photolytically generated carbonate radical and by UV light. PDI is prone to inactivation and aggregation by one-electron oxidants and UV light probably because of its high content of aromatic amino acids.

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90 percent protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.