Connecting structural and biochemical aspects of peroxiredoxins to cell signaling processes and proteic quality control

Abstract

Hydrogen peroxide and other peroxides are involved in cell signaling and therefore their levels are regulated in a quite sophisticated way. Among the various cellular systems capable of decomposing peroxides, 2-Cys peroxiredoxins (2-Cys Prx), which are peroxidases, utilize a highly reactive cysteine residue (peroxidase cysteine - CysP) to efficiently reduce a wide variety of hydroperoxides. The high reactivity associated with its great abundance are properties compatible with the evidence that 2-Cys Prx act as peroxide sensors in several cells, either directly or indirectly through interaction with signaling proteins (such as kinases and transcription factors) or by the oxidation of Thioredoxin (Trx). In fact, 2-Cys Prx are involved in cell signaling, being related in processes such as cancer, neurodegeneration, pathogen-host interaction, among others. However, the mechanisms underlying this phenomenon are not fully elucidated, in part because biochemical, kinetic and structural aspects related to 2-Cys Prxs are still unclear. Among other complicating aspects, 2-Cys Prx can undergo structural transitions between dimers, decamers or high molecular weight structures during the catalytic cycle, depending on the redox state of CysP among other factors. This structural variation has a direct influence on the processes of redox signaling and protein aggregation (which is related to several pathologies). We have contributed to clarify some of these aspects, having identified the role of specific amino acid residues. In the present project, the main objective is to investigate the impact of mutations in these amino acids residues of 2-Cys Prx in the cellular context. In this sense, we intend to use strains delta_tsa1, delta_tsa2, delta_tsa1/tsa2 among other yeast strains (Tsa1 and Tsa2 are two cytosolic Prx 2-Cys of Saccharomyces cerevisiae) expressing 2-Cys Prxs with mutations in residues previously evaluated in vitro, evaluating the oligomerization, CysP overoxidation, redox state of thioredoxins, besides effects on cell growth, susceptibility to hydroperoxides and aging among others. Alternatively, the CRISPR technique can be used to obtain the mutants. As complementary objectives, biochemical analyzes will be carried out to obtain the reduction kinetic constants of Tsa1 and mutants by Trx, as well as to carry out analysis of mutations equivalent to the cytosolic 2-Cys Prx of humans (Prx1 and Prx2). Finally, we intend to carry out structural analyzes by means of several techniques such as SAXS, cryo-electron microscopy, molecular exclusion chromatography, X-ray crystallography, to have a better understanding of how the mutations affect the structure of 2-Cys Prx. (AU)