Identification of three mutations in the Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene with familial amyotrophic lateral sclerosis: Transduction of human Cu,Zn-SOD into PC12 cells by HIV-1 TAT protein basic domain

Chih Ming Chou, Chang Jen Huang, Chwen Ming Shih, Yi Ping Chen, Tsang Pai Liu, Chien Tsu Chen

研究成果: 雜誌貢獻文章

12 引文 斯高帕斯(Scopus)

摘要

The most frequent genetic causes of amyotrophic lateral sclerosis (ALS) determined so far are mutations occurring in the gene coding for copper/zinc superoxide dismutase (Cu,Zn-SOD). The mechanism may involve the formation of hydroxyl radicals or malfunctioning of the SOD protein. Wild-type SOD1 was constructed into a transcription-translation expression vector to examine the SOD1 production in vitro. Wild-type SOD1 was highly expressed in Escherichia coli. Active SOD1 was expressed in a metal-dependent manner. To investigate the possible roles of genetic causes of ALS, a human Cu,Zn-SOD gene was fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain (RKKRRQRRR) of human immunodeficiency virus type 1 in a bacterial expression vector to produce a genetic in-frame Tat-SOD1 fusion protein. The expressed and purified Tat-SOD1 fusion proteins in E. coli can enter PC12 neural cells to observe the cellular consequences. Denatured Tat-SOD1 was successfully transduced into PC12 cells and retained its activity via protein refolding. Three point mutations, E21K, D90V, and D101G, were cloned by site-directed mutagenesis and showed lower SOD1 activity. In undifferentiated PC12 cells, wild-type Tat-SOD1 could prevent DNA fragmentation due to superoxide anion attacks generated by 35 mM paraquat, whereas mutant Tat-D101G enhanced cell death. Our results demonstrate that exogenous human Cu,Zn-SOD fused with Tat protein can be directly transduced into cells, and the delivered enzymatically active Tat-SOD exhibits a cellular protective function against oxidative stress.
原文英語
頁(從 - 到)303-313
頁數11
期刊Annals of the New York Academy of Sciences
1042
DOIs
出版狀態已發佈 - 2005

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • History and Philosophy of Science

指紋 深入研究「Identification of three mutations in the Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene with familial amyotrophic lateral sclerosis: Transduction of human Cu,Zn-SOD into PC12 cells by HIV-1 TAT protein basic domain」主題。共同形成了獨特的指紋。

  • 引用此