The Mechanism of Copper Toxicity to Iron Sulfur Proteins in Mitochondrial

Abstract: 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.

CSTR: 32200.14.cjcb.2016.07.0007