Leishmania major ascorbate peroxidase overexpression protects cells against reactive oxygen species-mediated cardiolipin oxidation.
Heme peroxidases are a class of multifunctional redox-active proteins found in all organisms. We recently cloned, expressed, and characterized an ascorbate peroxidase from Leishmania major (LmAPX) that was capable of detoxifying hydrogen peroxide. Localization studies using green fluorescent protein fusions revealed that LmAPX was localized within the mitochondria by its N-terminal signal sequence. Subcellular fractionation analysis of the cell homogenate by the Percoll density-gradient method and subsequent Western blot analysis with anti-LmAPX antibody further confirmed the mitochondrial localization of mature LmAPX. Submitochondrial fractionation analysis showed that the mature enzyme (~3.6 kDa shorter than the theoretical value of the whole gene) was present in the intermembrane space side of the inner membrane. Moreover, expression of the LmAPX gene was increased by treatment with exogenous H(2)O(2), indicating that LmAPX was induced by oxidative stress. To investigate the biological role of LmAPX we generated Leishmania cells overexpressing LmAPX in the mitochondria. Flow-cytometric analysis, thin-layer chromatography, and IC(50) measurements suggested that overexpression of LmAPX caused depletion of the mitochondrial ROS burden and conferred a protection against mitochondrial cardiolipin oxidation and increased tolerance to H(2)O(2). These results suggest that the single-copy LmAPX gene plays a protective role against oxidative damage.
Dolai
Subhankar
S
Division of Structural Biology and Bioinformatics, Indian Institute of Chemical Biology, Kolkata, India.
Yadav
Rajesh K
RK
Pal
Swati
S
Adak
Subrata
S
D000704:Analysis of Variance; D000818:Animals; D060387:Ascorbate Peroxidases; D015153:Blotting, Western; D002308:Cardiolipins / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006861:Hydrogen Peroxide / Q000378:metabolism; D020128:Inhibitory Concentration 50; D018320:Leishmania major / Q000166:cytology / Q000201:enzymology*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000378:metabolism / Q000648:ultrastructure; D015533:Transcriptional Activation; D015854:Up-Regulation
D002308:Cardiolipins; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases