De Souza 2017 Thesis
|de Souza LF (2017) Modulação redox de peroxirredoxinas e a participação dos sistemas da GSH e da Trx na proteção/função celular. Tese (doutorado) p103.|
Abstract: Thiol groups are amongst the main targets of reactive oxygen species (ERO), therefore they are at the center of all redox processes in the cell. Protein thiol oxidation can lead to loss of function, response signaling and cell death and, hence, the maintenance of thiols in cell is critical for cellular homeostasis. The glutathione (GSH) and thioredoxin (Trx) systems are the main redox buffers in cells. The continuous recycling of the oxidized forms of GSH and Trx is achieved by the action of glutathione reductase (GR) and thioredoxin reductase (TrxR), which keep the constant supply of electrons for the peroxidases. Among these, the peroxiredoxins (Prx) have a special role, acting as a peroxide sensor and marker of oxidative stress. The loss of redox homeostasis has been linked to several pathologies such as inflammation and cancer. Therefore, this work aims at study the importance of the GSH and Trx systems to cell protection against oxidants, and to analyze the redox modulation of Prx during inflammatory processes. In the first work, we investigated the effect of 2-acetylamino-3-[4-(2-acetylamino-2- carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), an inhibitor of GR and TrxR, on parameters of cell viability and redox balance in A172 glioblastoma cells. The inhibition of these enzymes drastically reduced cells' capacity to metabolized organic peroxides (but not H2O2) and, consequently, rendered cells more susceptible to organic peroxide. 2-AAPA also displayed a time and concentration dependent toxicity. We observed a marked oxidation of Prx 1, 2 and 3, as well as a severe mitochondrial dysfunction. Collectively, our results highlight the importance of GSH and Trx systems to cells' protection against oxidants. Also, we present evidence that oxidation of Prx and mitochondrial dysfunction are early events in 2-AAPA toxicity to A172 cells. In the second work, we studied the redox modulation of Prx during oxidative burst in differentiated promyelocytic HL60 cells. We demonstrated that the differentiation of these cells to a neutrophil-like phenotype with dimethylsulfoxide (DMSO) greatlydiminished Prx2 expression in a time-dependent manner, but didn´t change Prx1 expression. Pharmacological stimulation or exposure to microbes induced the oxidative burst in the differentiated cells, leading to Prx1 oxidation in a time-dependent manner. Oxidation of Prx1 was dependent on NADPH oxidase, because inhibition of this enzyme abolished this effect. Furthermore, inhibition of the oxidase by Diphenyleneiodonium chloride reduced the basal oxidation of Prx1 in unstimulated cells. Lastly, we discuss the presented results with data obtained from human neutrophils during my stay at the Center for Free Radical Research in New Zealand under supervision of Christine Winterbourn. We show for the first time that the Prx are completely oxidized under unstimulated conditions. Therefore, the presented results may have important implications for the role of Prx in inflammatory cells function. Overall, this thesis highlights the central role of the modulation of the redox environment to cell function and death, as well as shows, for the first time, a Prx fully oxidized in unstressed conditions.
• Keywords: Methylglyoxal, Glyoxalase, Glutathione reductase, Thioredoxin reductase, Glycation • Bioblast editor: Kandolf G
Labels: MiParea: Respiration
Stress:Oxidative stress;RONS Organism: Human Tissue;cell: Other cell lines Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ET