Abstract
BackgroundFerredoxin-NADP(H) reductase (FNR) from Pisum sativum and Flavodoxin (Fld) from Anabaena PCC 7119 have been reported to protect a variety of cells and organisms from oxidative insults. In this work, these two proteins were expressed in mitochondria of Cos-7 cells and tested for their efficacy to protect these cells from oxidative stress in vitro.Principal FindingsCos-7/pFNR and Cos-7/pFld cell lines expressing FNR and Fld, respectively, showed a significantly higher resistance to 24 h exposure to 300–600 µM hydrogen peroxide measured by LDH retention, MTT reduction, malondialdehyde (MDA) levels and lipid peroxide (LPO; FOX assay) levels. However, FNR and Fld did not exhibit any protection at shorter incubation times (2 h and 4 h) to 4 mM hydrogen peroxide or to a 48 h exposure to 300 µM methyl viologen. We found enhanced methyl viologen damage exerted by FNR that may be due to depletion of NADPH pools through NADPH-MV diaphorase activity as previously observed for other overexpressed enzymes.SignificanceThe results presented are a first report of antioxidant function of these heterologous enzymes of vegetal and cyanobacterial origin in mammalian cells.
Highlights
Ferredoxin-NADP(H) reductases (FNRs; EC 1.18.1.2) constitute a family of hydrophilic, monomeric enzymes that contain noncovalently bound flavin adenine dinucleotide (FAD) as a prosthetic group
In this work we investigated the capacity of P. sativum FNR and Anabaena Fld to protect Cos-7 cells from oxidative stress induced by hydrogen peroxide and methyl viologen
Previous work performed by various groups demonstrated the successful interactions and electron transfer of the components of hybrid systems as Anabaena FNR and bovine adrenodoxin [31] and bovine adrenodoxin reductase and Anabaena PCC 7119 Fld [32]
Summary
Ferredoxin-NADP(H) reductases (FNRs; EC 1.18.1.2) constitute a family of hydrophilic, monomeric enzymes that contain noncovalently bound flavin adenine dinucleotide (FAD) as a prosthetic group These ubiquitous flavoenzymes are present in animals, plants, parasites and prokaryotes, where they catalyze the reversible exchange of electrons between two molecules of a variety of obligatory one-electron carriers, such as ferredoxins (Fds) and flavodoxins (Flds), and a single molecule of NADP(H) [1]. Ferredoxin-NADP(H) reductase (FNR) from Pisum sativum and Flavodoxin (Fld) from Anabaena PCC 7119 have been reported to protect a variety of cells and organisms from oxidative insults. In this work, these two proteins were expressed in mitochondria of Cos-7 cells and tested for their efficacy to protect these cells from oxidative stress in vitro
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