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.
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.
Chou
Chih-Ming
CM
Department of Biochemistry, Taipei Medical University, Taipei, Taiwan.
Huang
Chang-Jen
CJ
Shih
Chwen-Ming
CM
Chen
Yi-Ping
YP
Liu
Tsang-Pai
TP
Chen
Chien-Tsu
CT
D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D002454:Cell Differentiation; D002470:Cell Survival; D019281:Dimerization; D004198:Disease Susceptibility; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D013481:Superoxides; D013482:Superoxide Dismutase