Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: mechanism and implication.
More than 400 million people are susceptible to oxidative stress due to glucose-6-phosphate dehydrogenase (G6PD) deficiency. Protein glutathionylation is believed to be responsible for loss of protein function and/or cellular signaling during oxidative stress. To elucidate the implications of G6PD deficiency specifically in cellular control of protein glutathionylation, we used hydroxyethyldisulfide (HEDS), an oxidant which undergoes disulfide exchange with existing thiols. G6PD deficient (E89) cells treated with HEDS showed a significant increase in protein glutathionylation compared to wild-type (K1) cells. In order to determine whether increase in global protein glutathionylation by HEDS leads to loss of function of an important protein, we compared the effect of HEDS on global protein glutathionylation with that of Ku protein function, a multifunctional DNA repair protein, using a novel ELISA. E89 cells treated with HEDS showed a significant loss of Ku protein binding to DNA. Cellular protein thiol and GSH, whose disulfide is involved in protein glutathionylation, were decreased by HEDS in E89 cells with no significant effect in K1 cells. E89 cells showed lower detoxification of HEDS, that is, conversion of disulfide HEDS to free sulfhydryl mercaptoethanol (ME), compared to K1 cells. K1 cells maintained their NADH level in the presence of HEDS but that of E89 cells decreased by tenfold following a similar exposure. NADPH, a cofactor required to maintain reduced form of the thiols, was decreased more in E89 than K1 cells. The specific role of G6PD in the control of such global protein glutathionylation and Ku function was further demonstrated by reintroducing the G6PD gene into E89 (A1A) cells, which showed a normal phenotype.
Ayene
Iraimoudi S
IS
Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA. ayenei@mlhs.org
Biaglow
John E
JE
Kachur
Alexander V
AV
Stamato
Thomas D
TD
Koch
Cameron J
CJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009249:NADP / Q000378:metabolism; D011485:Protein Binding
D009249:NADP; D004247:DNA; D005954:Glucosephosphate Dehydrogenase; D004265:DNA Helicases; D005978:Glutathione