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Eukaryotic Cell, November 2009, p. 1721-1731, Vol. 8, No. 11
1535-9778/09/$08.00+0     doi:10.1128/EC.00198-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Overexpression of Mitochondrial Leishmania major Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage {triangledown} ,{dagger}

Subhankar Dolai, Rajesh K. Yadav, Swati Pal, and Subrata Adak*

Division of Structural Biology & Bio-informatics, Indian Institute of Chemical Biology, Council of Scientific & Industrial Research, 4, Raja S.C. Mullick Road, Kolkata 700 032, India

Received 3 July 2009/ Accepted 31 August 2009

Ascorbate peroxidase from Leishmania major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H2O2 or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca2+ attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H2O2-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H2O2- or CPT-induced cytochrome c and endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H2O2 or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.

* Corresponding author. Mailing address: Division of Structural Biology & Bio-informatics, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India. Phone: 91 33 2473-6793. Fax: 91 33 2473-5197. E-mail: adaks{at}iicb.res.in

{triangledown} Published ahead of print on 11 September 2009.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

Eukaryotic Cell, November 2009, p. 1721-1731, Vol. 8, No. 11
1535-9778/09/$08.00+0     doi:10.1128/EC.00198-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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