Peroxynitrite preferentially oxidizes the dithiol redox motifs of protein-disulfide isomerase

Abstract

Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, such as cellular signaling and responses to cell-damaging events. PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants, and its dysfunction has been associated with several diseases in which oxidative stress plays a role. Because the kinetics and products of the reaction of these oxidants with PDI remain incompletely characterized, we investigated the reaction of PDI with the biological oxidant peroxynitrite. First, by determining the rate constant of the oxidation of PDI's redox-active Cys residues (Cys and Cys) by hydrogen peroxide ( = 17.3 ± 1.3 m s at pH 7.4 and 25 °C), we established that the measured decay of the intrinsic PDI fluorescence is appropriate for kinetic studies. The reaction of these PDI residues with peroxynitrite was considerably faster ( = (6.9 ± 0.2) × 10 m s), and both Cys residues were kinetically indistinguishable. Limited proteolysis, kinetic simulations, and MS analyses confirmed that peroxynitrite preferentially oxidizes the redox-active Cys residues of PDI to the corresponding sulfenic acids, which reacted with the resolving thiols at the active sites to produce disulfides ( Cys-Cys and Cys-Cys). A fraction of peroxynitrite, however, decayed to radicals that hydroxylated and nitrated other active-site residues (Trp, Trp, and Tyr). Excess peroxynitrite promoted further PDI oxidation, nitration, inactivation, and covalent oligomerization. We conclude that these PDI modifications may contribute to the pathogenic mechanism of several diseases associated with dysfunctional PDI.

The bicarbonate/carbon dioxide pair increases hydrogen peroxide-mediated hyperoxidation of human peroxiredoxin 1

“…This is likely due to the higher hydrophobicity of the tryptic peptides of Prx1 C P domain (retention time around 66 min under our experimental conditions) as compared with those of C R domain (retention time around 25 min under our experimental conditions). Indeed, hydrophobicity is a major factor influencing detection of a peptide by LC-MS/MS (37,41,42). Overall, the LC-MS/MS results (Figs.…”

“…Then, DTT (50 mM) was added to eliminate excess NEM (35). Following the removal of excess reagents, the samples were digested with trypsin and subjected to nano-ESI-Q-TOF-MS/MS analysis as described under "Experimental procedures" (36,37). The nano-ESI-Q-TOF-MS/MS analysis of the tryptic digests of all the Prx1 samples yielded sequence coverage of Ն 88% (data not shown).…”

Oxidação da proteína dissulfeto isomerase pelo hidroperóxido de urato e as implicações sobre o endotélio vascular

“…As amostras foram carregadas em coluna de nano-HPLC (ACQUITY UPLC BEH-C18, 20 mm x 180 µm, 5 µm, Waters, Milford, EUA) e eluídas a um fluxo de 400 nl/min, usando gradiente linear de 99 % de solvente A (água MilliQ contendo ácido fórmico 0,1 %) e 1 % de solvente B (acetonitrila contendo ácido fórmico 0,1 %), até (DOST et al, 2012;KAETZEL et al, 2004;PEIXOTO et al, 2018). Reação com hidroperóxido de urato promove agregação e inativação da proteína dissulfeto isomerase.…”

“…A agregação e inativação da PDI já foram verificadas após o tratamento da proteína com peroxinitrito. Nesse caso, o efeito é decorrente dos radicais derivados da decomposição desse oxidante, que parecem promover hidroxilação e nitração de resíduos de tirosina e triptofano próximos aos sítios redox ativos da PDI (PEIXOTO et al, 2018). Não seria o caso, claro, do HOOU.…”

Mecanismo associados à perda da regulação da nox1 NADPH oxidase pela dissulfeto isomerase proteica em células com ativação sustentada da via ras

“…does not have characteristics of a redox buffer, considering its peculiar redox properties and its compartmentalization (62,63). Moreover, the reactivity of PDIA1 towards hydrogen peroxide is quite slow (64), indicating that PDIA1 is unlikely to be a mass-effect sensor of oxidant state such as the peroxiredoxins. Rather, PDIs appear to locally target more specific protein clients (30).…”