Biochemical and structural characterization of peroxiredoxins from Aspergillus fumigatus, human opportunistic pathogen

Author(s)

Renata Bannitz Fernandes

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2019-03-27

Examining board members

Luis Eduardo Soares Netto; Madia Trujillo Garré; Sayuri Miyamoto; Daniela Ramos Truzzi

• Advisor: Luis Eduardo Soares Netto

Abstract

Peroxiredoxins (Prxs) are highly efficient peroxidases that depend on a catalytic triad composed of Thr/Ser, Cys and Arg residues. These enzymes can be classified as 2-Cys Prx and 1-Cys Prx, according to the number of Cys residues involved in catalysis or according to structural characteristics that divide the Prxs in 6 subfamilies. The Prx6 subfamily is almost exclusively composed by 1-Cys Prx enzymes. This subfamily is still poorly characterized and the identities of their biological reductants are still controversial. Some of the reductant candidates are thioredoxin (Trx) and ascorbate. The mammalian members of this subfamily possess an additional phospholipase A2 (PLA2) activity that relies on another catalytic triad that is composed by His, Ser and Asp residues. In this thesis, we investigated biochemical and structural aspects of two enzymes belonging to the Prx6 subfamily from Aspergillus fumigatus: the cytosolic AfPrx1 and the mitochondrial AfPrxC. A. fumigatus is the most important pathogenic fungus transmitted through the air. Initially, we characterized the oxidation of AfPrx1 and AfPrxC by H2O2, t-BOOH, CuOOH, LAOOH and ONOO-, monitoring redox dependent changes in the intrinsic fluorescence of these enzymes. Additionally, we characterized the reduction of AfPrx1 and AfPrxC by Trx, Grx, ascorbate, ergothioneine, GSH and H2S. Interestingly, only H2S reduced these proteins efficiently κAfPrx1 ≈ 103 M-1 s-1 and κk;AfPrxC ≈ 104 M-1 s-1). In addition to the peroxidase activity, we determined for the first time the phospholipase activity (PLA2) for a non-mammalian Prx, using liposomes radioactive-labeled. AfPrx1 and AfPrxC displayed PLA2 activity (≈ 200 nmol/ h/ mg of protein) that was inhibited by MJ33 (about 75 %) and enhanced after phosphorylation by MAPK. Moreover, we also showed that these proteins were important for fungus survival during studies co-cultures with macrophages. Furthermore, AfPrx1 was important to detoxify A. fumigatus from exogenous added H2O2 as observed through an electrochemical approach. The biochemical and structural characterization of these Prxs described herein can open new therapeutic strategies, since at least AfPrx1 is involved in fungus virulence in a mouse model (AU)

• FAPESP's process: 13/14955-2 - Functional and structural characterization of AfPrxA, a thiol peroxidase from the human pathogen fungus Aspergillus fumigatus and a potential target for drug development
• Grantee: Renata Bannitz Fernandes
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)