| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
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.
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