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 researcher: 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

Abstract

S-nitrosation of cysteine residues is believed to be a universal and common nitric oxide (NO*) dependent post-translational modification that affects the activity, association, and cell compartmentalization 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 nitrosyl 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 their appearance and concentration coincide with cellulars-nitrosoprotein formation and content. It has been suggested that s-nitrosoproteins are formed because of the nucleophilic attack of a cysteine residue to the NO group bound to LIP in DNICs. Such mechanism is attractive in 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 the 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 proteins 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 the 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.(AU)