The primary objective of this proposal is to gain insight into functions of Yb-SC proteins in intracellular steroid and carcinogen binding mechanisms. The studies are based on our initial observations that these proteins, that were originally categorized as a subclass of glutathione-S-transferases, exhibit select high affinity binding capacity for steroids and certain polycyclic aromatic hydrocarbons, and may be converted to DNA binding forms by a temperature dependent release of bound GSH. The Yb-SC proteins therefore represent high affinity high capacity binding components in many mammalian tissues that have the potential to govern accessibility of steroids for interaction with receptors and other cellular macromolecules. Molecular mechanisms of interaction of steroid hormones and polycyclic aromatic hydrocarbons, will be analyzed with homogenous preparations of Yb-SC. Spectroscopic, kinetic and equilibrium binding methods will be developed for rapid and reliable screening of compounds that interact with these proteins in vitro, in cell extracts, and in vivo, to determine specificity, affinity, as well as structural requirements for binding of these substances. Structural aspects of the proteins will be probed especially with regard to the two distinct binding domains; a primary site involved in binding of glucocorticoids, progestins and androgens and a secondary region for binding of estrogens and anti-estrogens which is also the catalytic site. Nuclear forms of Yb-SC will be studied in the context of the transport of steroids and carcinogens to the nucleus. The proteins will be studied in cell culture systems with several different cell lines and steroid or carcinogen resistant variants of these, to explore possible functions of Yb-SC in steroid hormone action and metabolism. Tissue specificity, sex differences and factors that regulate synthesis and levels of Yb-SC will be analyzed and these will be correlated to known biological responses elicited by steroids, """"""""anti-steroids"""""""" and chemical carcinogens.
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