Study of the interaction of dinitrosyl iron complexes (DNIC) and thioredoxins

• Grant number: 22/11354-7
• Support Opportunities: Scholarships in Brazil - Master
• Start date: September 01, 2022
• End date: August 31, 2024
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Daniela Ramos Truzzi
• Grantee: Arthur Migliatti Vetorazzi Ferreira de Pinho
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 19/17483-0 - Dinitrosyl iron complexes (DNICs): formation, reactivity, and implications for the physiopathology of nitric oxide, AP.JP

Abstract

Thioredoxin (Trx), a ubiquitous protein in organisms, exhibits a highly conserved active site in which two cysteines are separated in the primary structure only by two amino acid residues (CXXC). These cysteine residues can be in the oxidized (disulfide, S-S) or the reduced state (thiols, SH) and enable the Trx function in hydrogen peroxide (H2O2) metabolism and a diversity of interactions with other proteins. Trx also participates in nitric oxide (NO) metabolism, since it is capable of denitrosating nitrosothiols. The interplay between NO and H2O2 cellular metabolism is also evidenced by experiments showing that an increase in NO concentration leads to a reduction in H2O2 metabolism via mechanisms that are not yet elucidated. In this context, it is important to highlight that NO gives rise to several metabolites in the biological medium, among which the dinitrosyl iron complexes (DNICs) are the most abundant. DNICs are mainly bonded to thiol proteins, which can coordinate in mono or bidentate form. Initial kinetic studies in our Laboratory indicate that DNICs containing bidentate ligands are more stable in aqueous medium pH 7.4 and thus should be favored in cells. Therefore, Trx could be a target for the formation of biological DNICs, since it enables bidentate coordination through both cysteine residues in the active site. Also, the bond of Trx to DNIC (DNIC-Trx) could change this enzyme catalytic activity and explain the decrease in H2O2 metabolism as NO concentration rises. In fact, initial studies in our group showed that S. cerevisiae thioredoxin 1(Trx1) is capable of bonding to DNICs in a fast way in the aqueous medium, leading to a reduction in the enzyme's activity. Based on that, this project aims to study the formation of DNICs containing Trx1 of S. cerevisiae (DNIC-Trx1) as ligand, calculate rate constants, and quantify the reduction in the enzyme's activity due to the DNIC-Trx1 formation. Then, the activity of Trx1 will be measured in RAW 264.7 macrophages under conditions that favor or not the intracellular DNICs formation. The results of this study can contribute to the identification of proteins bonded to biological DNICs and elucidate the mechanisms responsible for reducing H2O2 metabolism as a result of NO concentration. (AU)