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该文探讨了铜对线粒体内铁硫蛋白的毒性机理。通过包装慢病毒将Hep G2细胞中铜转运蛋白ATP7B(ATPase copper transporting beta)基因敲低,并用铜离子处理构建高铜细胞模型。通过免疫印迹、胶内酶活、紫外–可见光分光光度法检测细胞线粒体内铁硫蛋白、非铁硫蛋白及铁硫簇组装蛋白量和活性的改变;用电镜观察高铜模型中线粒体的形态改变;用海马能量代谢分析仪检测铜离子对细胞能量代谢的影响。结果发现,高铜细胞模型线粒体内铁硫簇组装蛋白ISCA2(ironsulfur cluster assembly 2)及ISCU(iron-sulfur cluster assembly enzyme)水平下降,抑制了铁硫簇的组装,并进一步影响了线粒体内[2Fe-2S]型及[4Fe-4S]型铁硫蛋白功能,但并不影响非铁硫蛋白。高铜状态也影响了呼吸链复合体活性及线粒体能量代谢,并导致线粒体形态发生改变。这些结果表明,异常累积的铜离子也会通过抑制线粒体中铁硫簇的组装,影响线粒体内铁硫蛋白的功能。
This article explores the toxicity of copper to mitochondrial iron-sulfur protein. The packaging of lentivirus knockdown the ATP7b (ATPase copper transporting beta) gene in Hep G2 cells and the treatment with copper ion to construct a high copper cell model. The changes of mitochondrial contents of iron-sulfur protein, non-iron-sulfur protein and iron-sulfur cluster in the mitochondria were detected by immunoblotting, in-gel enzyme and UV-Vis spectrophotometry. Morphological changes of mitochondria in high copper The effect of copper ion on cellular energy metabolism was detected by hippocampal energy metabolism analyzer. The results showed that the level of iron-sulfur cluster assembly enzyme (ISCA2) and iron-sulfur cluster assembly enzyme (ISCU) in the mitochondria of high-copper cell model decreased, which inhibited the assembly of iron-sulfur clusters and further affected the [2Fe -2S] and [4Fe-4S] -type iron-sulfur protein function, but does not affect the non-iron-sulfur protein. High copper status also affects respiratory chain complex activity and mitochondrial energy metabolism, and leads to altered mitochondrial morphology. These results indicate that abnormally accumulated copper ions also affect the function of iron-sulfur protein in mitochondria by inhibiting the assembly of iron-sulfur clusters in the mitochondria.