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Mechanism and kinetic of s-nitrosoprotein formation from dinitrosyl iron complexes associated to glutathione s-transferases (GST-DNIC)

Grant number: 15/04942-6
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): June 01, 2015
Effective date (End): May 31, 2016
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:José Carlos Toledo Junior
Grantee:Laura Carneiro da Silva
Home Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID


S-nitrosation of cystein residues is believed to be a universal and common nitric oxide (NO*) dependent post translational modification that affects the activity, association and cell compartimentalization of numerous proteins and enzymes and controls or modulates distinct biological processes such as metabolism, gene expression and cell death. However, there exists certain resistance to the full acceptance of s-nitrosation as a signaling mechanism. It still is not clear how protein s-nitrosation occurs in cells and whether the mechanism is coherent with principles of biological communication such as rapidity, selectivity and reversibility. It is clear that there is no direct reaction between NO* and thiols, thus s-nitrosation requires intermediacy of species reactive toward NO*. Recent reports point to the possible involvement of the labile iron pool (LIP) and cellular dinitrosyl iron complexes (DNICs), the products of the NO* and LIP rapid reaction. DNICs are protein associated and ubiquitously formed in cells exposed to endogenous or exogenous NO* and its appearance and concentration coincide with cellular s-nitrosoprotein formation and content. It has been suggested that s-nitrosoproteins are formed as a result of the nucleophilic attack of a cysteine residue to the NO group bound to LIP in DNICs. Such mechanism is attractive on the context of signaling through s-nitrosation since it could be selective [governed by specific protein(DNIC)/protein(thiol) interaction] and possibly rapid and catalytic, although this has not been characterized so far. Our main goal in this proposal is to study the kinetics and the mechanism of protein(DNIC)/protein(thiol) reaction that leads to protein thiol nitrosation. To this end, as the DNIC source we will use glutathione s-transferase protein DNICs, which is one of the few cellular protein known to carry DNICs in cells. As nitrosation targets we have selected mammals Prx II and Prx VI proteins because they both have reactive and low pKa thiols. The study will contribute to the elucidation of mechanism and kinetics of s-nitrosoprotein formation and hopefully will allow better control of s-nitrosation events in cells, benefiting studies of NO* induced biological processes known to be transduced through s-nitrosation.