An antisense oligonucleotide to 1-cys peroxiredoxin causes lipid peroxidation and apoptosis in lung epithelial cells
An antisense oligonucleotide to 1-cys peroxiredoxin causes lipid peroxidation and apoptosis in lung epithelial cells
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DOI：
10.1074/jbc.m204222200
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发表时间：
2002-12-20
影响因子：
4.8
通讯作者：
Fisher, AB
中科院分区：
文献类型：
--
作者：
Pak, JH;Manevich, Y;Fisher, AB
1-半胱氨酸过氧化物氧还蛋白（1-cysPrx）是过氧化物氧还蛋白超家族的一员，可还原磷脂氢过氧化物以及有机过氧化物和H2 O2。为了确定1-cysPrx的生理功能，我们使用反义策略来抑制L2细胞（大鼠肺上皮细胞系）中的内源性1-cysPrx。设计了一种25个碱基的反义吗啉代寡核苷酸，以结合与大鼠1-cysPrx mRNA中的翻译起始位点（-18至+7）重叠的互补序列，阻断蛋白质合成。与随机寡核苷酸处理和对照（仅溶剂）细胞相比，用反义寡核苷酸处理48 h导致1-cysPrx蛋白含量抑制约60%（通过免疫印迹分析测定），谷胱甘肽过氧化物酶活性降低约44%。通过高压液相色谱法测定共轭二烯（反义为260 pMol/10（6）个细胞，随机寡核苷酸和对照细胞为70 pMol/10（6）个细胞）和二苯基-1-芘基膦（脂质过氧化的探针）的荧光，证明磷脂酰胆碱氢过氧化物在质膜中的蓄积。膜联蛋白V和丙啶染色阳性的细胞百分比。反义处理后28 h碘含量为40%，48 h为80%。TdT介导的dUTP缺口末端标记法在48小时表明，在反义处理的细胞，被阻断的腺病毒编码1-cysPrx或预处理与维生素E类似物的感染前的DNA片段化。结果表明，1-cysPrx可以在完整的细胞中作为抗氧化酶发挥作用，以减少磷脂氢过氧化物的积累，并防止凋亡细胞死亡。
1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, reduces phospholipid hydroperoxides as well as organic peroxides and H2O2. To determine the physiological function(s) of 1-cysPrx, we have used an antisense strategy to suppress endogenous 1-cysPrx in L2 cells, a rat lung epithelial cell line. A 25-base antisense morpholino oligonucleotide was designed to bind a complementary sequence overlapping the translational start site (-18 to +7) in the rat 1-cysPrx mRNA, blocking protein synthesis. Treatment with an antisense oligonucleotide for 48 h resulted in approximately 60% suppression of the 1-cysPrx protein content as measured by immunoblot analysis and an approximately 44% decrease of glutathione peroxidase activity as compared with random oligonucleotide treated and control (vehicle only) cells. Accumulation of phosphatidylcholine hydroperoxide in plasma membranes was demonstrated by high pressure liquid chromatography assay for conjugated dienes (260 pMol/10(6) cells for antisense versus 70 pmol/10(6) cells for random oligonucleotide and control cells) and by fluorescence of diphenyl-1-pyrenylphosphine, a probe for lipid peroxidation. The percentage of cells showing positive staining for annexin V and propidium. iodide after antisense treatment was 40% at 28 h and 80% at 48 h. TdT-mediated dUTP nick end labeling assay at 48 h indicated DNA fragmentation in antisense-treated cells that was blocked by prior infection with adenovirus encoding 1-cysPrx or by pretreatment with a vitamin E analogue. The results indicate that 1-cysPrx can function in the intact cell as an antioxidant enzyme to reduce the accumulation of phospholipid hydroperoxides and prevent apoptotic cell death.