Kinetic characterization of the reduction of 1-cys peroxiredoxins by ascorbate.

• Grant number: 12/00629-3
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: April 01, 2012
• End date: February 28, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Luis Eduardo Soares Netto
• Grantee: Valesca Anschau
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Peroxiredoxins (Prx) are ubiquitous enzymes capable of degrading organic and hydrogen peroxide as well as peroxynitrite. They are classified as 1-Cys- Prxs or 2-Cys, depending on the number of cysteines involved in its catalytic cycle. In the case of most 1-Cys Prxs, the identity of biological reducing agent is still controversial. One possibility raised by our group is that ascorbate could support the peroxidase activity of 1-Cys Prx (Monteiro e col., 2007). In this proposal, we intend to get more insights on the relevance of this pathway in biological systems. Accordingly, two strategies will be pursued: (1) determination of enzymatic parameters related to the reduction of 1-Cys Prx by ascorbate, and (2) investigate the response of lung cells to oxidative stress under conditions where the levels 1-Cys prx and / or ascorbate are inhibited. Regarding the first strategy, some in silico modeling and site directed mutations have already been performed in our laboratory (using the Rattus norvegicus Prdx6 as a model of 1-Cys Prx) and indicated the involvement of some amino acids in the reduction of 1 - Cys Prx by ascorbate. However, to proceed in the understanding of the biological relevance of this pathway is essential to quantify the reduction of 1-Cys peroxiredoxin by ascorbate. To achieve this goal, we intend to use of electrodes for hydrogen peroxide (Free Radical Analyzer 4100 from World Precision Instruments) and also by peroxixantone, a fluorescent probe based on boron that react with hydrogen peroxide producing a fluorescent product. Preliminary results indicated that the two methodologies do not suffer interference by ascorbate. Regarding the strategy (2), we intend to silence the levels of 1-Cys prx (Prdx6 in mammals) by using a procedure already established in the literature. The ascorbate levels are modulated in accordance with the addition of this vitamin in the culture medium of human cells. This control of the levels of ascorbate will be possible because humans are unable to synthesize ascorbate. Thus, separately or simultaneously inhibiting the levels of ascorbate and 1-Cys prx we can comparatively analyze the response of human lung cells to oxidative stress induced by peroxides.

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

ANSCHAU, Valesca. Kinetic characterization of the reduction of 1-Cys Peroxiredoxins by ascorbate. 2017. Doctoral Thesis - Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB) São Paulo.