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Investigation of structural transitions and reactivity towards TSA1 (thiol specific antioxidant protein 1) peroxides of Saccharomyces cerevisiae

Grant number: 10/00172-8
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): August 01, 2010
Effective date (End): January 31, 2014
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Marcos Antonio de Oliveira
Grantee:Carlos Abrunhosa Tairum Junior
Home Institution: Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil

Abstract

Thiol specific antioxidant (TSA) or Thioredoxin peroxidase (TPX) constitute a group of antioxidant proteins that have been widely studied for his performance in the decomposition of various types of peroxides, such as lipid peroxides, peroxynitrite and hydrogen peroxide (H2O2). As well as many thiol oxireductases, TPxs are common in nature; however, unlike the others, they are found in various cell compartments. Five isoforms have been identified In Saccharomyces cerevisiae: three found in the cytoplasm (Tsa1, Tsa2 and Ahp1), one in mitochondria (Prx1) and one in nucleus (nTPx). Among this yeast Tpx, Tsa1 and Tsa2 call attention, since they present great similarity in the primary structure (86% identity and 96% similarity), but differ considerably in terms of the number of copies in the cell (Tsa1 ~ 378,000 molecules; Tsa2 ~ 4,820) and on the pKa of the catalytic cysteine, which probably can be explained by small differences in the intrinsic structural characteristics of enzymes. The ability of decomposition of peroxides by Tsa is related to the presence in their active sites of a cysteine residue of high reactivity, called cysteine peroxidatic (CysP), directly involved in decomposition of these substrates. Additionally, the vast majority of Tsa has a second cysteine, called cysteine resolution (CysR), which forms a disulfide with CysP during the course of redox cycle. Most TPx uses Thioredoxin (Trx) - a low molecular weight protein with two vicinal cysteines - as a reducing substrate during the catalytic cycle. During the redox cycling reactions, Tsa needs to execute a structural change in the helix in which reactive cysteine residue is found, alternating between two conformations: fully folded (FF), when protein is in the reduced form and ±-helix in which CysP is located is completely tangled, and locally unfolded (LU), observed after interaction with the peroxide, which activates the unfolding of the ±-helix for the formation of disulfide between the cysteines. We recently demonstrated that amino acid residues E50 and R146 have a key role in FF/LU transition states, since Tsa1 mutants of S. cerevisiae (Tsa1E50A and Tsa1R146Q) present relevant structural changes and loss of peroxidase activity front of Trx. This research project aims to investigate the molecular mechanisms responsible for structural changes and the Tsa1 FF/LU transition states. To this will be carried out crystallization experiments and determination of three-dimensional structure of mutants Tsa1E50A and Tsa1R146Q through the methodology of crystallography X-ray. It will also be effectuated the mutation Tsa1D141A and its effect will be evaluated by in vitro enzyme activity, size exclusion chromatography and circular dichroism spectroscopy. Additionally, to study the differences in cysteine peroxidase pKa and reactivity for organic peroxides and H2O2 of Tsa1 and Tsa2, it will be made substitutions of amino acids Ile39, Thr44 and Pro143 of Tsa1 to residues found in Tsa2 in corresponding spacial position. After, structural and functional effects of these mutations (Tsa1I39V, Tsa1T44S and Tsa1P143S) will be assessed to verify which ones are important in differentiating of Tsa1 and Tsa2. Once Tsa1 S. cerevisiae has representative orthologs in mammals - in particular humans -results of this work may contribute to a better understanding of the behavior of these proteins that are related to genetic diseases involving protein aggregation and uncontrolled cell growth, such as Alzheimer's and cancer. (AU)

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