Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation

Abstract

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.

Structural characterization of the trypsinized estrogen receptor

“…All our studies were performed in the presence of 10 mM mercaptoethanol or 1 mM DTT, but a buried disulfide bridge may not have been accessible to the solvent for reduction by these agents. Others have shown that reduced sulfhydryl groups in the steroid binding domain of the glucocorticoid receptor are essential for the maintenance of high-affinity hormone binding (Bresnick et al, 1988;Meshinchi et al, 1990). However, in our studies with the ER, a disulfide bridge (the oxidized form of the sulfhydryl group) would be necessary to bind the 10-kDa fragment to the other fragments and thereby maintain the steroid binding domain's structure and function in solution.…”

Interaction of hsp90 with steroid receptors: organizing some diverse observations and presenting the newest concepts

“…It is known that in cytosols containing extensive protease activity (e.g. rat liver cytosol) molybdate partially stabilizes the glucocorticoid receptor to degradation (Bresnick et al, 1988). As all of the other effects of molybdate on steroid receptors can be attributed to stabilization of receptor-hsp90 complexes, we have suggested that binding to hsp90 stabilizes the receptor to proteolysis in cytosol.…”

A Redox Mechanism Controls Differential DNA Binding Activities of Hypoxia-inducible Factor (HIF) 1α and the HIF-like Factor

“…Moreover, nuclear translocation of the GR showed varied sensitivities to H 2 O 2 in different cell types, with GR activity on reporter genes being affected by physiological concentrations of H 2 O 2 in mammary tumor cells (53), indicating that GR function can be influenced by endogenous redox factors and varying natural redox states according to cell type. Whereas low concentrations of H 2 O 2 (0.5-2 mM) can disrupt nuclear localization of the GR, considerably higher concentrations (20 -100 mM) are required to disrupt ligand binding in vitro (54), implying that separate receptor activities are subject to differential redox control. As HIF-1␣ has recently been shown to undergo hypoxia-induced nuclear translocation (55), it will be interesting to investigate whether this process is influenced by redox conditions.…”