Purchase this article with an account.
J.E. Sears, G. Hoppe; Glutaredoxin Reduces Chaperone Activity via Deglutathionylation of Hsc70 . Invest. Ophthalmol. Vis. Sci. 2004;45(13):743.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Protein S–glutathionylation is a reversible modification that regulates protein structure and function. We have previously reported that in cell–free assays oxidative stress induces S–glutathionylation of Hsc70 (Hsc70–SG) and increases the chaperone activity of this protein. We hypothesized that modification of Hsc70 by glutathione occurs in intact human cultured RPE cells, and that Hsc70–SG is a substrate of glutaredoxin–1 (grx–1). Methods: 2D gel electrophoresis and western blot analysis were used to prove that Hsc70 becomes glutathionylated in cultured cells treated with 0.25 mM diamide. Eighteen µM Hsc70 was treated with 5 mM GSSG followed by incubation with either 0.15, 1.5, or 3 µM reduced grx–1 prior to analysis in a luciferase denaturation assay. Results: Hsc70 was resolved by 2D gel electrophoresis and proved to be glutathionylated in intact cells after oxidative stress. Hsc70–SG was five fold more active in preventing luciferase aggregation. Reduced grx–1 caused a dose–dependent decrease in chaperone activity. Paradoxically, the lowest dose grx–1 tested was synergistic with Hsc70–SG. Conclusions: A subfraction of Hsc70/Hsp70 is glutathionylated after oxidative stress in intact cells. Glutathione–modified Hsc70 is more active than reduced Hsc70 and is a substrate of grx–1. Oxidoreductases catalyze reversible thiol/disulfide exchange in heat shock proteins and provide a general paradigm of regulation of protein structure and function in response to oxidation.
This PDF is available to Subscribers Only