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Investigation of the molecular determinants involved in the interaction with substrates of Tsa1 and Tsa2 of Saccharomyces cerevisiae

Grant number: 11/13500-6
Support Opportunities:Scholarships in Brazil - Doctorate
Effective date (Start): October 01, 2011
Effective date (End): September 30, 2015
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal Investigator:Marcos Antonio de Oliveira
Grantee:Carlos Alexandre Breyer
Host Institution: Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil

Abstract

Peroxiredoxins (Prx) are antioxidant proteins able to decompose a wide variety of hydroperoxides that have stood out because of its catalytic efficiency, high abundance and distribution in different cellular compartments. The Prx are able to reduce their substrates using a highly reactive cysteine residue located in its active site, called cysteine peroxidase (CP-SH), which in the process of decomposition of peroxides is oxidized to cysteine sulfenic acid (CP-SOH) .In Saccharomyces cerevisiae, there are two isoforms of cytosolic Prx (Tsa1 and Tsa2), which have great similarity (86% identity and 96% similarity). In addition to peroxidase activity, it has been shown that Tsa1 and Tsa2 have chaperone activity. However, the intracellular concentration of these proteins varies significantly. While Tsa1 has ~ 378 000 molecules per cell, Tsa2 has only 4800 in log phase of growth. Thus, it has been postulated that despite the great similarity, Tsa1 is involved mainly in cellular defense, in response to thermal or oxidative insults. Meanwhile Tsa2 would have a greater involvement in signal transduction, in events such as growth and apoptosis. It has been demonstrated that both enzymes can be reduced by cytosolic thioredoxin (Trx1 and Trx2). Just as the cytosolic Prx, Trx1 and Trx2 share high similarity in their primary structures (78% identity and 89% similarity) and are often considered redundant proteins. However, no study has yet experimented to see if the rates of reduction of Tsa1 and Tsa2 were equivalent when using Trx1 or Trx2. The structure of Tsa1 was recently determined. Its analysis reveals that 27 of the 28 amino acids that differ from Tsa2 are on the surface of the molecule, suggesting specific interactions with cytosolic Trx. This project aims to carry out systematic analysis of the molecular relationship of Tsa1and Tsa2 with Trx1 and Trx2, which should result in a greater understanding of the processes of reduction/superoxide of Tsa1 and Tsa2, and thereby to the cellular processes in which these proteins participate. To this end, we intend to determine the structure of Tsa2 in the reduced state and oxidized to disulfide for understanding in detail their differences with Tsa1 and molecular relationships with their substrates. We will also make the comparative evaluation of the reduction rates of Tsa1 and Tsa2 by Trx1 or Trx2 through the NADPH oxidation assay. The reduction rates decisively influence the superoxide, and oligomerization of chaperone function of Tsa1 and Tsa2. They will be evaluated by western blot (WB) using antibodies specific to super-oxidized forms (in vitro and in vivo), molecular exclusion chromatography (SEC ) and dynamic light scattering (DLS). For further developments of the research, mutations/combinations of amino acids in Tsa2 targeting specific sites of the interaction surface of Tsa/Trx will be conducted. Those mutations will replace amino acids in Tsa2 by their correspondents that occupy the same position in Tsa1 (Tsa2 (S45T), Tsa2 (K97N) and Tsa2 (D150N).

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Scientific publications (7)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DE MOURA, WERNER ALFINITO FEIO; SCHULTZ, LEONARDO; BREYER, CARLOS ALEXANDRE; DE OLIVEIRA, ANA LAURA PIRES; TAIRUM, CARLOS ABRUNHOSA; FERNANDES, GABRIELLA COSTA; TOYAMA, MARCOS HIKARI; PESSOA-JR, ADALBERTO; MONTEIRO, GISELE; DE OLIVEIRA, MARCOS ANTONIO. Functional and structural evaluation of the antileukaemic enzymel-asparaginase II expressed at low temperature by differentEscherichia colistrains. Biotechnology Letters, . (13/08617-7, 17/19942-7, 17/25272-4, 19/04054-4, 14/22039-9, 16/19245-1, 17/20291-0, 11/13500-6)
DE OLIVEIRA, MARCOS ANTONIO; TAIRUM, CARLOS A.; SOARES NETTO, LUIS EDUARDO; PIRES DE OLIVEIRA, ANA LAURA; ALEIXO-SILVA, ROGERIO LUIS; MONTANHERO CABRERA, VITORIA ISABELA; BREYER, CARLOS A.; DOS SANTOS, MELINA CARDOSO. Relevance of peroxiredoxins in pathogenic microorganisms. Applied Microbiology and Biotechnology, v. 105, n. 14-15, . (20/00845-4, 17/06263-4, 13/07937-8, 20/02868-1, 16/15849-0, 11/13500-6, 17/19942-7, 19/04054-4)
ROMANELLO, KAREN S.; TEIXEIRA, KARINA K. L.; SILVA, JOAO PEDRO M. O.; NAGAMATSU, SHEILA T.; BEZERRA, MARCOS ANDRE C.; DOMINGOS, IGOR F.; MARTINE, DIEGO A. P.; ARAUJO, ADERSON S.; LANARO, CAROLINA; BREYER, CARLOS A.; et al. Global analysis of erythroid cells redox status reveals the involvement of Prdx1 and Prdx2 in the severity of beta thalassemia. PLoS One, v. 13, n. 12, . (11/13500-6)
TAIRUM, CARLOS A.; SANTOS, MELINA CARDOSO; BREYER, CARLOS ALEXANDRE; PIRES DE OLIVEIRA, ANA LAURA; MONTANHERO CABRERA, VITORIA ISABELA; TOLEDO-SILVA, GUILHERME; MORI, GUSTAVO MARUYAMA; TOYAMA, MARCOS HIKARI; SOARES NETTO, LUIS EDUARDO; DE OLIVEIRA, MARCOS ANTONIO. Effects of Serine or Threonine in the Active Site of Typical 2-Cys Prx on Hyperoxidation Susceptibility and on Chaperone Activity. ANTIOXIDANTS, v. 10, n. 7, . (20/02868-1, 19/04054-4, 17/20291-0, 17/19942-7, 18/12628-8, 11/13500-6, 17/06263-4, 13/07937-8)
DE PAULA, CARLA PERES; DOS SANTOS, MELINA CARDOSO; TAIRUM, CARLOS A.; BREYER, CARLOS ALEXANDRE; TOLEDO-SILVA, GUILHERME; TOYAMA, MARCOS HIKARI; MORI, GUSTAVO MARUYAMA; DE OLIVEIRA, MARCOS ANTONIO. Glutaredoxin-like protein (GLP)-a novel bacteria sulfurtransferase that protects cells against cyanide and oxidative stresses. Applied Microbiology and Biotechnology, v. 104, n. 12, . (13/16192-6, 13/07937-8, 17/19942-7, 17/06263-4, 11/13500-6, 17/20291-0, 10/00172-8, 10/16827-3)
DE MOURA, WERNER ALFINITO FEIO; SCHULTZ, LEONARDO; BREYER, CARLOS ALEXANDRE; DE OLIVEIRA, ANA LAURA PIRES; TAIRUM, CARLOS ABRUNHOSA; FERNANDES, GABRIELLA COSTA; TOYAMA, MARCOS HIKARI; PESSOA-JR, ADALBERTO; MONTEIRO, GISELE; DE OLIVEIRA, MARCOS ANTONIO. Functional and structural evaluation of the antileukaemic enzymel-asparaginase II expressed at low temperature by differentEscherichia colistrains. Biotechnology Letters, v. 42, n. 11, p. 12-pg., . (13/08617-7, 11/13500-6, 16/19245-1, 17/25272-4, 19/04054-4, 17/19942-7, 14/22039-9, 17/20291-0)
GUILHEN LONGO, LARISSA VALLE; BREYER, CARLOS ALEXANDRE; NOVAES, GABRIELA MACHADO; GEGEMBAUER, GREGORY; LEITAO JR, NATANAEL PINHEIRO; OCTAVIANO, CARLA ELIZABETE; TOYAMA, MARCOS HIKARI; DE OLIVEIRA, MARCOS ANTONIO; PUCCIA, ROSANA. The Human PathogenParacoccidioides brasiliensisHas a Unique 1-Cys Peroxiredoxin That Localizes Both Intracellularly and at the Cell Surface. FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, v. 10, . (13/07937-8, 17/19942-7, 06/05095-6, 13/25950-1, 17/20291-0, 11/13500-6)

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