The mechanism of cell damage by mutant superoxide dismutase 1 of human amyotrophic lateral sclerosis

我们将野生型和突变型(G37R、G85R、G93A、S134N)超氧化物歧化酶1基因亚克隆到两个表达载体pcDNA3和PEF-BOS上。将SOD1基因分别导入COS-7细胞。转染后48h，COS7细胞在PEF-BOS中可见突变型SOD1蛋白聚集在核周，具有较高的启动子活性。这些聚集体定位于内质网(ER)。聚集的形成与线粒体和微管蛋白在这些细胞中的异常分布有关。将突变的SOD1基因亚克隆到启动子活性相对较低的pcDNA3中，在COS 7细胞中不产生任何聚集体。然而，在培养上清液中加入蛋白酶体抑制剂lactacystin，可以诱导突变蛋白在pcDNA3中的SOD1突变基因细胞中聚集。在pc DNA3和pEF-BOS中，未观察到SOD1基因的聚集。结论：突变的SOD1蛋白的过度表达超过了蛋白酶体的降解能力，导致了突变蛋白在内质网中的聚集。这些聚集体可能导致细胞内细胞器功能障碍，如线粒体。

We subcloned wild and four mutant (G37R, G85R, G93A, S134N) superoxide dismutase 1 (SOD1) genes to two expression vectors, pcDNA3 and pEF-BOS. Each SOD1 gene was transfected to COS 7 cells. Forty eight hours after transfection, aggregates of the mutant SOD1 proteins were observed in the perikarya of COS7 cells transfected with any mutant SOD1 gene in pEF-BOS, which has a high promoter activity. These aggregates were localized to endoplasmic reticulum (ER). The aggregate formation was associated with abnormal distribution of both mitochondria and tubulin in these cells. The mutant SOD1 genes subcloned to pcDNA3, which has a relatively low promotor activity, did not produce any aggregates in COS 7 cells. However, the administration of lactacystin, a proteasome inhibitor, to culture medium induced the aggregation of the mutant proteins in cells transfected with the mutant SOD1 genes in pcDNA3. No aggregation was observed in cells transfected with wild SOD1 gene in pcDNA3 or pEF-BOS.In conclusion, it is suggested that the over-expression of mutant SOD1 proteins beyond the capacity of degradation by proteasome results in the aggregation of the mutant proteins in ER. These aggregates may induce a dysfunction of intracellular organelles, such as mitochondria.