Overexpression of Mitochondrial Leishmania major Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage
Overexpression of Mitochondrial Leishmania major Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage
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DOI：
10.1128/ec.00198-09
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发表时间：
2009-11-01
期刊：
影响因子：
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通讯作者：
Adak, Subrata
中科院分区：
文献类型：
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作者：
Dolai, Subhankar;Yadav, Rajesh K.;Adak, Subrata
来自利什曼原虫的抗坏血酸过氧化物酶（LmAPX）是清除线粒体呼吸链中活性氧的关键酶之一。我们研究了线粒体LmAPX是否在氧化应激诱导的细胞凋亡中起作用。细胞匀浆中还原性谷胱甘肽（GSH）和蛋白羰基含量的测定表明，LmAPX的过表达保护利什曼原虫细胞免受H2O2或喜树碱（CPT）处理下GSH的消耗和蛋白质的氧化损伤。共聚焦显微镜和荧光光谱数据显示，lmapx过表达细胞获得的细胞内Ca2+升高始终低于对照细胞。流式细胞术分析数据和共聚焦显微镜观察强烈表明，LmAPX过表达保护细胞免受h2o2诱导的线粒体膜去极化和ATP减少。Western blot数据表明，LmAPX的过表达可以阻止H2O2-或cpt诱导的细胞色素c和内切酶G从线粒体释放并随后在细胞质中积累。流式细胞术Caspase活性测定显示，凋亡刺激下lmapx过表达细胞的Caspase样蛋白酶活性降低。细胞表面暴露的磷脂酰丝氨酸数据和DNA片段分析结果表明，LmAPX的过表达使利什曼原虫细胞更能抵抗H2O2或CPT处理引起的凋亡。综上所述，这些结果表明，LmAPX在L. major线粒体中的组成性过表达可以防止细胞受到氧化应激的有害影响，即线粒体功能障碍和细胞死亡。
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.