铜对线粒体中铁硫蛋白功能的毒性机制研究  

The Mechanism of Copper Toxicity to Iron Sulfur Proteins in Mitochondrial

作　　者：李艳纯[1] 杜璟[1] 刘若兰[1] 任雪营 林楚贤 李江辉[1] 谭国强[1] 吕建新[1] 

机构地区：[1]温州医科大学检验医学院、生命科学学院,检验医学教育部重点实验室,浙江省医学遗传学重点实验室,温州325035

出　　处：《中国细胞生物学学报》2016年第7期811-820,共10页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:81500440);国家高技术研究发展计划(863计划)(批准号:2014AA06A514)资助的课题~~

摘　　要：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 study discussed the toxicity of copper to iron sulfur proteins in the mitochondria. We conducted the RNA-mediated knockdown of ATP7B gene which involved in the export of copper out of the cells by lentivirus infection and established the high copper cell model by adding copper ions in the medium of ATP7B knockdown HepG2 cells. We detected the activity changes of iron sulfur proteins and mitochondria complexes by in-gel activity assay or spectrophotometry. Western blot was used to detect the protein levels of iron sulfur cluster assembly protein and other mitochondrial proteins without iron sulfur cluster. And the mitochondrial morphology of the copper treated HepG2 was observed by transmission electron microscopy. At last, the oxygen consumption rates （OCRs） of cultured HepG2 cells treated with and without copper ions was analyzed by the Seahorse Bioscience XF96 Extracellular Flux Analyzer. The results showed that the iron sulfur cluster assembly 2 （ISCA2） and iron-sulfur cluster assembly enzyme （ISCU） were decreased in the mitochondrial of high copper cell model, which inhibiting the assembly of iron sulfur clusters, and affecting the function of iron sulfur proteins with [2Fe-2S] or [4Fe-4S]. But copper treated cells had no effect on other mitochondrial proteins without iron sulfur cluster such as isocitrate dehydrogenase 2 （IDH2） and malate dehydrogenase 2 （MDH2）. Moreover, high copper status also affected the activity of respiratory chain complexes and cell energy metabolism, leading to the changes of mitochondrial morphology and mitochondrial membrane potential. These results suggested that abnormal accumulation of copper ions could inhibit the iron sulfur cluster assembly and affect the function of iron sulfur proteins in mitochondrial.

关 键 词：铜毒性 铁硫簇 铁硫蛋白 能量代谢 

分 类 号：R114[医药卫生—卫生毒理学]