| Grant number: | 16/12248-5 |
| Support Opportunities: | Scholarships abroad - Research Internship - Doctorate (Direct) |
| Start date: | September 12, 2016 |
| End date: | February 12, 2017 |
| Field of knowledge: | Biological Sciences - Biochemistry - Molecular Biology |
| Principal Investigator: | Luis Eduardo Soares Netto |
| Grantee: | Renata Bannitz Fernandes |
| Supervisor: | Aron Baer Fisher |
| Host Institution: | Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil |
| Institution abroad: | University of Pennsylvania, United States |
| Associated to the scholarship: | 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, BP.DD |
Abstract Peroxiredoxins (Prxs) are Cys-based thiol-dependent peroxidases that contain one (1-Cys Prx) or two (2-Cys Prx) Cys residues involved in catalysis. To date, all characterized Prxs display the universal motif PxxxT/SxxC and can reduce hydroperoxides with extraordinary rates, ranging from 106 to 108 M-1 s-1. In spite of being a heterogeneous family, all Prxs present the peroxidase activity dependent on Thr-Cys-Arg catalytic triad. For mammalian enzymes belonging to the Prx6 subfamily (mainly 1-Cys Prx), an additional acidic Ca2+-independent phospholipase A2 activity (aiPLA2) was previously described and was associated with other residues, in this case Ser-Asp-His catalytic triad. This activity is mainly involved in lung surfactant metabolism and membrane remodeling and is maximum at acid pH, compatible with the lysosomal localization. Aspergillus fumigatus is a human pathogen fungus that possesses three Prxs belonging to the subfamily Prx6 that are named herein as AfPrxA, AfPrxB and AfPrxC. We had already shown (unpublished results) that these three Prxs display high peroxidase activities related with H2O2 reduction (107 M-1 s-1). Now, we aim to verify if these three Prxs from A. fumigatus present the additional aiPLA2 activity in this BEPE proposal. In silico analyses suggest that AfPrxB and AfPrxC possess the conserved catalytic triad typical for the aiPLA2 activity. In addition, they also display the target lysosomal peptide highly conserved compared to the mammal peptide previously determined. The aiPLA2 activity will be measured with the recombinant proteins purified and incubated with radiolabeled unilamellar liposomes with different phospholipid compositions in a calcium-free buffer. Furthermore, we aim to investigate this activity in fungus extracts from wild type and mutant strains using different inhibitors and also investigate the role of the PLA2 activity catalytic serine and the peroxidase catalytic cysteine by site-direct mutagenesis. Therefore, the identification and characterization of aiPLA2 activities may provide more insights on the physiological roles of these enzymes. (AU) | |
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