The heat-stable cytosolic factor that promotes glucocorticoid receptor binding to DNA is neither thioredoxin nor ribonuclease

Abstract

Treatment of rat liver cytosol containing temperature-transformed [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). However, transformed receptors that are treated with MMTS and then separated from low Mr components of cytosol by passage through a column of Sephadex G-50 have very little DNA-binding activity when DTT is added to regenerate sulfhydryl moities. The receptors will bind to DNA if whole liver cytosol or boiled liver cytosol is added in addition to DTT. The effect of boiled cytosol is mimicked by purified rat thioredoxin or bovine RNase A in a manner that does not reflect the reducing activity of the former or the catalytic activity of the latter. This suggests that the reported ability of each of these heat-stable peptides to stimulate DNA binding by glucocorticoid receptors is not a biologically relevant action. We suggest that stimulation of DNA binding of partially purified receptors by boiled cytosol does not constitute a reconstitution of a complete cytosolic system in which the dissociated receptor must associate with a specific heat-stable accessory protein required for DNA binding, as has been suggested in the "two-step" model of receptor transformation recently proposed by Schmidt et al. (Schmidt T.J., Miller-Diener, A., Webb M.L. and Litwack G. (1985) J. biol. Chem. 260, 16255-16262).

Glucocorticoid receptor binding to calf thymus DNA. 1. Identification and characterization of a macromolecular factor involved in receptor-steroid complex binding to DNA

“…Several other factors have been described that increase the DNA binding of activated glucocorticoid complexes. All of these factors are larger than our factor [i.e., ~72K (Payvar & Wrange, 1987), 70K (Tymoczko et al, 1988), 10-12K (Grippo et al, 1985;Tienrungroj et al, 1987b), and >5K (Schmidt et al, 1985;Harmon et al, 1988)], and none were isolated from crude nuclei. Small molecular weight substances, like /3-mercaptoethanol and Zn2+ (Sabbah et al, 1987), that increase the DNA binding of activated complexes cannot be our factor since these compounds would be included in Sephadex G-10 columns and since added thiols did not cause increased DNA binding (Table III).…”

“…Copyright. Published 1990 by the American Chemical Society been previously observed by us (Simons, 1977) and others (Tienrungroj et al, 1987b) that, under some conditions, more activated receptor-steroid complexes bind to nuclei than to DNA. The initial purpose of the present study, therefore, was to resolve the differences between nuclear and DNA binding of activated glucocorticoid complexes.…”

“…Nonspecific DNA facilitates the binding of trans-acting factors to specific sequences (Berg et al, 1982). Furthermore, without nonspecific DNA binding to act as a buffer, activated complex binding to a small number of high-affinity SREs would become saturated, and would cause full induction or repression of responsive genes, over a very narrow concentration of steroid (Winter & von Hippie, 1981). Partially for these reasons, and due to the ready availability and ease of experimental manipulations, nonspecific DNA (particularly DNA-cellulose) has been used in most studies of activation.…”

Control of Trx1 redox state modulates protection against methyl methanesulfonate-induced DNA damage via stabilization of p21

“…Indeed, protection of Trx1 against MMS-induced DNA damage could have been involved in DNA repair as redox signalling has been shown to affect APE1/Ref1 function, an apurinic/apyrimidinic endonuclease in the DNA base excision repair pathway (38,39), and APE1/Ref1 was shown to regulate the p53/p21 system (37,40). In addition, it was reported that the heat-stable cytosolic factor that promotes glucocorticoid receptor binding to DNA after MMS treatment is neither thioredoxin nor ribonuclease (41), suggesting that Trx1-mediated protection against MMS-induced DNA damage might be specific through certain cellular factors.…”

Identification of vitamin D2 metabolites and age-related changes in the 1,25-dihydroxyvitamin D3 steroid receptor in male Fischer 344 rats

“…This would imply that an inhibitor to DNA binding is present in the cytosol of aged rats. Alternatively, one cannot dismiss the possibllty that an enhancer to binding exists in the young cytosols(38,84).A series of mixing experiments examining whether or not this putative inhibitor could negatively influence DNA binding in other vitamin D receptor preparations would be useful. Secondly, the detection of two protein bands at 52K and 50K spe cific for the kidney receptor was unexpected.…”