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Singlet molecular oxygen (1 O2 ) is generated in biological systems and reacts with different biomolecules. Proteins are a major target for 1 O2 , and His, Tyr, Met, Cys, and Trp are oxidized at physiological pH. In the present study, the modification of lysozyme protein by 1 O2 was investigated using mass spectrometry approaches. The experimental findings showed methionine, histidine, and tryptophan oxidation. The experiments were achieved using [18 O]-labeled 1 O2 released from thermolabile endoperoxides in association with nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry. The structural characterization by nLC-MS/MS of the amino acids in the tryptic peptides of the proteins showed addition of [18 O]-labeling atoms in different amino acids.

Singlet molecular oxygen (1 O2 ) has been associated with a number of physiological processes. Despite the recognized importance of 1 O2 -mediated protein modifications, little is known about the role of this oxidant in crosslink formation and protein aggregation. Thus, using lysozyme as a model, the present study sought to investigate the involvement of 1 O2 in crosslink formation. Lysozyme was photochemically oxidized in the presence of rose bengal or chemically oxidized using [18 O]-labeled 1 O2 released from thermolabile endoperoxides. It was concluded that both 1 O2 generating systems induce lysozyme crosslinking and aggregation. Using SDS-PAGE and nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry, the results clearly demonstrated that 1 O2 is directly involved in the formation of covalent crosslinks involving the amino acids histidine, lysine, and tryptophan.

Apoptosis-inducing factor (AIF) is a flavoprotein and essential partner of the CHCHD4 redox protein during the mitochondrial intermembrane space import machinery. Mammalian AIF has three cysteine residues, which have received little attention. Previous reports have evidenced a redox interaction between AIF and thioredoxin 1 (Trx1), particularly after oxidant conditions. Therefore, we asked whether the cysteine residues of the human AIF could be oxidized. Our data showed that endogenous AIF could be oxidized to disulfide-linked conjugates (DLC). Overexpressed WT AIF in HEK293T cells, as well as recombinant WT AIF, formed DLC. Expression of C256S, C317S or C441S AIF mutants severely inhibited DLC formation in cells exposed to oxidants. In vitro, DLC formation was completely precluded with C256S and C441S AIF mutants and partially inhibited with the C317S mutant. DLC was shown to enhance cellular susceptibility to apoptosis induced by staurosporine, likely by preventing AIF to maintain mitochondrial oxidative phosphorylation. Cells with decreased expression of Trx1 produced more AIF DLC than those with normal Trx1 levels, and in vitro, Trx1 was able to decrease the amount of AIF DLC. Finally, confocal analysis, as well as immunoblotting of mitochondrial fraction, indicated that a fraction of Trx1 is present in mitochondria. Overall, these data provide evidence that all three cysteine residues of AIF can be oxidized to DLC, which can be disrupted by mitochondrial Trx1.

O oxigênio molecular singlete (1O2) é formado em sistemas biológicos e reage com diferentes biomoléculas. Proteínas representam um dos principais alvos de oxidação, devido as suas altas concentrações em organismos. Em pH fisiológico 1O2 reage com His, Tyr, Met, Cys e Trp. Neste trabalho investigamos a oxidação causada pelo 1O2 e a formação de dimerização em uma proteína modelo, a lisozima. A identificação dos principais produtos de oxidação e dimerização foi realizada por sequenciamento de peptídeos através de nano cromatografia acoplada a espectrometria de massas (nLC-MS/MS). A geração de 1O2 foi realizada por fotossensibilização utilizando luz e rosa bengala como fotossensibilizador, e pela decomposição térmica de endoperóxidos derivado do naftaleno DHPN16O2 e DHPN18O2, uma fonte limpa de 1O2 no meio reacional. Os resultados demonstraram que a reação do oxigênio singlete com lisozima acarreta oxidação dos resíduos de Met, His e Trp. A caracterização da estrutura primária por nLC-MS/MS dos aminoácidos confirmou a adição de átomos de oxigênio marcado (18O). A lisozima é constituída apenas de um resíduo de histidina (His15) e as oxidações identificadas foram adições de massas de +14 Da (descrita como 2-oxo-histidina), +16 e +32 Da. Os resíduos de metionina (Met12 e Met105) foram identificados como sulfóxidos (MetSO - adição de massas de +16 Da). Para os resíduos de triptofano foram identificados a formação de quinurenina (adição de massas de +4 Da), +16 e +32 Da. As oxidações levaram a formação de dimerização na proteína caracterizada por eletroforese em gel e nLC-MS/MS. O objetivo principal do trabalho foi analisar a ligação cruzada entre o resíduo de histidina 2-oxohistidina na lisozima. Entretanto, foram identificadas ligações cruzadas entre 2- oxo-histidina e resíduos de lisina, além de ligações cruzadas com resíduos de triptofano oxidado. Em consequência dos resultados obtidos com a proteína modelo, avaliamos as oxidações e formação de dímeros em proteínas extraídas do cristalino do olho bovino. Diferentes tipos de modificações foram observados, além da formação de dímeros entre resíduos de histidina (2-oxoHis-His) caracterizados por nLC-MS/MS e bioinformática. Os dados obtidos neste trabalho, fornecem evidências da ocorrência simultânea de formação de ligações cruzadas entre diferentes proteicas após exposição a 1O2. O trabalho resultou na identificação e sequenciamento através de nLC-MS/MS de peptídeos oxidados por 1O2 a partir de uma proteína modelo. Esses resultados reiteram o importante papel do 1O2 em reações com proteínas além do seu envolvimento no desenvolvimento de condições patológicas. A dimerização formada na ligação cruzada em 2-oxo-His-His representa um possível novo biomarcador para o 1O2 em sistemas biológicos Singlet molecular oxygen (1O2) can be generated in biological systems, reacting with different biomolecules. Proteins are major target for oxidants due to higher concentration in organisms. At physiological pH, 1O2 may react with the following aminoacids: His, Tyr, Met, Cys and Trp. Here, we investigated oxidation and dimerization reactions of proteins exposed to 1O2 using lysozyme as a model. Modifications of lysozyme by 1O2 were investigated using mass spectrometry approaches. Identification of the main oxidation and dimerization products were performed by peptide sequencing by nano-chromatography coupled to mass spectrometry (nLC-MS/MS). Singlet oxygen was generated using visible light and rose Bengal as photosensitizer, and from the decomposition of thermolabile endoperoxides DHPN16O2 e DHPN18O2, clean sources of 1O2. Experimental findings showed oxidation of Met, His, and Trp residuesin lysozyme. Structural characterization by nLC-MS/MS of the oxidative modifications in lysozyme tryptic peptides showed the addition of [18O]-labeled atoms in different amino acid residues. Lysozyme has in its structure a single histidine residue (His15). We identified shifts of +14 Da (described as oxohistidine), +16 and +32 Da in this residue. Methionine residues (Met12 and Met105) were oxidized to sulfoxides (MetSO mass shift of +16 Da). Modifications in tryptophan residues were identified as kynurenine (shift mass of +4 Da), +16 and + 32 Da. Oxidized lysozyme subjected to SDS-Page showed dimmers formation. The main aim was to analyze cross-linking formation between 2-oxo-histidine residues in lysozyme. However, cross-links between 2- oxo-histidine and lysine residues, and cross-links between oxidized tryptophan residues have been identified. Following results obtained with the protein model, we evaluated oxidation and the dimers formation in proteins extracted from the lens of the bovine eye. Analysis performed in nLC-MS/MS and bioinformatics identified different types of modifications, including formation of dimers with histidine residues (2-oxo-His-His). The data provided evidence for simultaneous occurrence of protein cross-linking on exposure 1O2. These results demonstrated the important role of 1O2 in protein reactions beyond its involvement in developing of pathological conditions. In conclusion, dimerization of proteins through 2-oxo-His residues may be a possible new biomarker for 1O2 in biological systems.

Singlet molecular oxygen (

The present chapter focuses on how the use of [18O]-labeled endoperoxides and hydroperoxides can be applied to study mechanistic aspects related to singlet molecular oxygen (1O2) generation and reactions in biological system. Identification of 1O2, which was achieved using different approaches: [18O]-labeled molecular oxygen, a clean naphthalene derivative [18O]-labeled thermolabile endoperoxide as a source of [18O]-labeled 1O2, chemical trapping of [18O]-1O2 with anthracene derivatives and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The peroxidation reactions of the main cellular targets including nucleic acids, proteins and unsaturated lipids have received major attention during the last two decades. We highlighted some key mechanistic aspects related to [18O]-peroxides decomposition to alkoxyl or peroxyl radicals. As a biochemical application this chapter focused on the recombination reactions involving lipid-derived [18O]-labeled peroxyl radicals that can be formed on different lipid species giving rise to [18O]-labeled 1O2 through the mechanism proposed by Russell. A relevant major topic deals with the search for the molecular signature of the 1O2 formation in targeted biomolecules within cells. It may be anticipated that labeled peroxides and [18O]-labeled 1O2 in association with a sensitive mass spectrometric method such as LC-ESI-MS/MS should constitute powerful tools for this purpose.

Este trabalho objetiva verificar os potenciais das plantas Trichilia pallida e Sesamum indicum na prospecção de acaricidas botânicos, para serem utilizados como nova alternativa para controle do ixodídeo Rhipicephalus sanguineus. O extrato hexânico (e.h.) de folhas de T. pallida e o óleo essencial de sementes de S. indicum foram obtidos em laboratório, a partir de amostras coletadas, respectivamente, em uma área de Mata Atlântica e em fazendas de cultivo no Brasil. Larvas de R. sanguineus foram obtidas a partir de fêmeas ingurgitadas, coletadas em ambientes urbanos naturalmente infestados. Os bioensaios foram realizados em quadruplicata, a 27 ± 1oC, RH ? 80% e fotoperíodo de 12 horas. Cerca de 50 larvas, de 14 a 21 dias de idade, foram acondicionadas em envelopes de papel-filtro, impregnados com diferentes concentrações das substâncias vegetais, obtidas a partir da diluição de soluções-padrão. Em cada bioensaio, foram utilizados quatro envelopes impregnados com cada concentração testada. O grupo controle usou a mesma quantidade de larvas, submetidas a: 1. Envelopes sem nenhum tratamento; 2. Envelopes tratados com água destilada e, 3. Envelopes com solventes utilizados na preparação de soluções vegetais. Pela ação do e.h. de folhas de T. pallida e do óleo de S. indicum, após 24 horas de exposição, obtiveram-se, respectivamente, CL50 de 4.660 ppm e 107.729 ppm e CL99 de 14.217 ppm e 279.912 ppm. Após 48h, obtiveram-se, respectivamente, CL50 de 1.555 ppm e 78.880 ppm e CL99 de 3.431 ppm e 221.255 ppm. Mortalidade larval significativa não foi observada no grupo controle (P < 0,05). This paper aims at verifying the potentials of Trichilia pallida and Sesamum indicum in the prospection of botanical acaricide, to be used as a new alternative to R. sanguineus control. Hexanic extract (h.e.) of T. pallida leaves and essential oil from seeds of S. indicum were obtained in laboratory, from samples collected, respectively, in an area of Atlantic forest and in agricultural farms, in Brazil. R. sanguineus larvae were obtained from engorged females collected in naturally infested urban environs. Bioassays were carried out in quadruplicate, at 27 ± 1oC, RH ? 80% and photoperiod of 12 hours. About 50 larvae, from 14 to 21 days of age, were conditioned in filter paper envelopes, impregnated with different concentrations of vegetable substances, obtained from dilution of stock solutions. In each bioassay, four envelopes impregnated with each tested concentration were used. The control group used the same amount of larvae, submitted to: 1. envelopes with no treatment; 2. envelopes with distilled water and, 3. envelopes having solvents used in the preparation of vegetable solutions. For the action of h.e. leaves of T. pallida and S. indicum oil, after 24h of exposure, it was obtained, respectively, LC50 of 4,660 ppm and 107,729 ppm and LC99 of 14,217 ppm and 279,912 ppm. After 48h, it was obtained, respectively, LC50 of 1,555 ppm and 78,880 ppm and LC99 of 3,431 ppm and 221,255 ppm. There was no significant mortality rate within the control group (P < 0.05). Made available in DSpace on 2019-11-21T18:08:30Z (GMT). No. of bitstreams: 1 26907.pdf: 229077 bytes, checksum: 7a95db95b8292ea0f42eea7f00e9bd3b (MD5) Previous issue date: 2019