Structural and Biochemical Characterization of Antioxidant Enzymes AfPrx1 and AfPrxC from Aspergillus fumigatus

• Grant number: 24/07167-2
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: June 01, 2024
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Luis Eduardo Soares Netto
• Grantee: Maria Tereza Oliveira Batista
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

Aspergillus fumigatus is a mycopathogen capable of causing invasive pulmonaryaspergillosis (IPA), a severe systemic lung infection with high mortality rates. Successfulinfections by A. fumigatus require the fungus to cope with oxidative stress imposed bythe host. Peroxiredoxins (Prxs) are abundant thiol peroxidases crucial for decomposingperoxides, playing a vital role in pathogen detoxification and response to host-generatedoxidative stress. Collaborating with Dr. Iran Malavazi's group (UFSCAR), wedemonstrated that AfPrx1 is involved in the virulence of this fungus, possibly through thereduction of hydroperoxides, including H2O2 generated by the host. Another A. fumigatusPrx, AfPrxC, is localized in mitochondria, while AfPrx1 is in the cytosol. Both AfPrx1and AfPrxC are 1-Cys Prxs, relying on a single cysteine residue for catalytic activity. Dra.Renata Fernandes characterized the biochemical and structural aspects of AfPrx1 andAfPrxC during her doctoral studies, revealing that both proteins form dimers throughvarious techniques. Renata also characterized the oxidation kinetics of these 1-Cys Prxsby different hydroperoxides. However, a comprehensive understanding of these enzymes'action mechanisms requires elucidating the atomic-resolution tertiary structure andidentifying biological reducers.Therefore, the aim of this project is to structurally and biochemically characterize theantioxidant enzymes AfPrx1 and AfPrxC from the human pathogen Aspergillusfumigatus. We are investigating the reduction of sulfenic acid (Cys-SOH) in these 1-CysPrxs by ascorbate using the DCPIP (2,6-dichlorophenolindophenol) competition method.Preliminary results indicate that AfPrx1 is reduced by ascorbate at pH 7.4, with a secondorderrate constant of 4.9 x 102 M-1.s-1. Simultaneously, crystallization assays for AfPrx1and AfPrxC, with well-established expression and purification protocols yielding highlysoluble recombinant proteins, are being conducted. Ultimately, we intend to obtainstructures of AfPrx1 and AfPrxC complexed with ascorbate (or erythroascorbate) throughsoaking or co-crystallization.In a later phase, we plan to analyze peroxide levels and D-erythroascorbate derivatives (a5-carbon analogue of ascorbate) in A. fumigatus strains with disrupted AfPrx1 andAfPrxC genes. H2O2 levels can be analyzed in real-time and non-invasively in A.fumigatus using genetically encoded probes targeted to different cellular compartmentssuch as the cytosol and mitochondria. Strains deficient in erythroascorbate synthesis willalso be generated. As redox processes are involved in A. fumigatus interactions with hosts,the information generated in this project may contribute to understanding the virulencemechanisms of this pathogenic fungus.