[The effects of cysteines on the function of human glutathion S-transferase pi(GSTp) under cell oxidative stress].
Site-directed mutagenesis was used to generate three cysteine mutants of GSTp, C(47/101), C(14/47/101) and C(14/47/101/169). GSTp, C(47/101), C(14/47/101) and C(14/47/101/169) were transfected into 293 cells separately and GST activity was determined by using CDNB as substrate. Data showed that each cysteine mutant inhibited endogenous GST catalyzatic activity and had remarkable dominant negative effect. The expression vectors of wide type GSTp and its cysteine mutants were co-transfected with c-Jun, NF-kappaB, or p21 luciferase reporting vector, into 293 cells separately, luciferase activity showed that C(14/47/101) and C(14/47/101/169) can dramatically activate c-Jun and p21 transcriptional activity. Each cysteine mutant can increase endogenous p21 level, and also increased mortality rate of 293 cells when exposed to H2O2. These results suggest that cysteine residues of GSTp play an important role in protecting cells against oxitative stress.
Sima
Jian
J
College of Life Science, Nanjing Normal University, Nanjing 210097, China.
He
Lan
L
Zhu
Jian
J
Xue
Bin
B
Tai
Yi Lin
YL
Zhang
Shuang Quan
SQ
Yin
Zhi Min
ZM
D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000502:physiology*; D051549:Glutathione S-Transferase pi / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D018384:Oxidative Stress / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D003545:Cysteine