Two-hybrid yeast test system for assessment of estrogenic activity of chemical compounds

Abstract

The search for natural and synthetic compounds exhibiting estrogenic and antiestrogenic activity is a topical problem of modern biology and medicine. Compounds exhibiting estrogenic activity are widely used in medical practice to correct functioning and to treat a number of endocrine disorders and sexual diseases. Antagonists of steroid hormones may be used for designing antitumor preparation to treat hormonedependent malignancies. In addition, the search for compounds that display estrogenic activity and the determination of their concentrations in various water bodies and soil are important for assessing environmental pollution with mimetics of steroid hormones, which affect metabolic pathways and mimic or modify the effect of estrogens and, as a result, can disturb the functions of the endocrine system and pose a threat to health of humans and animals.It is known that the main target for estrogen and estrogen-like compounds is the nuclear steroid receptor (ER receptor), which binds in a specific manner with the steroid hormone and regulates gene expression [1]. In human and homeothermic animals, inactivated ER receptors exist in large complexes, being associated with heat shock proteins. After estrogen binds with the ER receptor, heat shock proteins dissociate, thereby triggering a conformational change that activates the receptor and causes its dimerization. The homodimeric complex displays a high affinity for the specific DNA sequence located in the regulatory region of estrogeninducible genes. Binding with this region initiates the biosynthesis of transcriptional factors that, in turn, activate the target gene expression. Then, mRNA is translated to yield protein that are the final effectors of cell responses observed. By inducing the synthesis of new proteins affecting cell functions, estrogens highly effectively influence various biochemical and physiological processes. Xenoestrogens or mimetics may function as ER ligands and bind with receptors, thus modulating endocrine metabolic pathways via the receptor-mediated process.One of the key problems in search for and study of compounds exhibiting steroid hormone activity is designing new test systems for rapid, highly sensitive, and highly selective assessment of the hormonal activity of preparations of interest. This study was devoted to designing a two-hybrid yeast test system containing human estrogen receptors and β -galactosidase gene as a reporter enzyme. For this purpose, we used the plasmids pCL1, pGBKT7-Lam (Clontech, United States); two expression plasmids ( pGBT9-hER α , containing the ER α human estrogen receptor gene, and pGAD424-hTIF2, containing the TIF2 expression coactivator gene) were kindly provided by Dr. Nishihara Tsutomu (Department of Environmental Bioscience, Osaka University, Japan). The Saccharomyces cerevisiae strain Y187 ( MAT α , 112 , gal4 ∆ , met -, gal80 ∆ , URA3::GAL1 UAS -GAL1 TATA -lacZ ) from Clontech (United States) was used as yeast culture. Yeast cells were transformed with plasmids using the lithium-acetate method; t...

Estrogenic activity of triterpene glycosides in yeast two-hybrid assay

“…In this study we used the yeast two-hybrid assay with the estrogen receptor, hER␣, and the co-activator, TIF2, by the method described in a previous paper [35]. Briefly, we introduced two expression plasmids, pGBT9-hER␣ and pGAAD424-TIF2, kindly provided by Dr. Nishihara Tsutomu (Department of Environmental Bioscience, Osaka University, Japan), into yeast cells (Saccharomyces cerevisiae Y190) by the lithium acetate method according to the smallscale yeast transformation procedure (CLONTECH, Yeast Protocols Handbook).…”