The steroid hormone levels and receptors of steroid hormone receptors in rat muscles during physical exertion

It is shown that the testosterone content in skeletal muscles of female albino rats is 2-fold decreased, while the estradiol content-1.5-fold increased and progesteron content showed no changes after systematic physical exercises. The pharmacokinetic investigations showed that androgen half-life in the organism decreased from 8 to 5 h under physical exercises. The amount of androgen receptors in cytosol of skeletal muscles increases from 1.34 +/- 0.08 to 1.71 +/- 0.10 fmol/mg per 1 mg of protein. Kd is 0.40 +/- 0.03 and 0.48 +/- 0.08 nM, respectively. Sensitivity of the organism to hormonal signal play an important role in the metabolism regulation in skeletal muscles along with the hormonal content change during the organism adaptation to physical exercises.     

Creation of a pluripotent ubiquitin-conjugating enzyme

We describe the creation of a pluripotent ubiquitin-conjugating enzyme (E2) generated through a single amino acid substitution within the catalytic domain of RAD6 (UBC2). This RAD6 derivative carries out the stress-related function of UBC4 and the cell cycle function of CDC34 while maintaining its own DNA repair function. Furthermore, it carries out CDC34's function in the absence of the CDC34 carboxy-terminal extension. By using sequence and structural comparisons, the residues that define the unique functions of these three E2s were found on the E2 catalytic face partitioned to either side by a conserved divide. One of these patches corresponds to a binding site for both HECT and RING domain proteins, suggesting that a single substitution in the catalytic domain of RAD6 confers upon it the ability to interact with multiple ubiquitin protein ligases (E3s). Other amino acid substitutions made within the catalytic domain of RAD6 either caused loss of its DNA repair function or modified its ability to carry out multiple E2 functions. These observations suggest that while HECT and RING domain binding may generally be localized to a specific patch on the E2 surface, other regions of the functional E2 face also play a role in specificity. Finally, these data also indicate that RAD6 uses a different functional region than either UBC4 or CDC34, allowing it to acquire the functions of these E2s while maintaining its own. The pluripotent RAD6 derivative, coupled with sequence, structural, and phylogenetic data, suggests that E2s have diverged from a common multifunctional progenitor.     

Intracellular traffic of steroid hormone receptors

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. It is a complex signal. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between the nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. The same phenomenon exists for estradiol and glucocorticoid receptors. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement into the cytoplasm is not understood. We have grafted different nuclear localization signals (NLSs) onto β-galactosidase and have studied the traffic of this protein using heterokaryons and microinjection experiments. We have demonstrated that the same NLSs are involved in both the inward and the outward movement of proteins through the nuclear membrane. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally. Using these techniques, we have shown that two major antiprogestins, RU486 and ZK98299, act at the same distal level of hormone action.     

Huntington's disease and steroid hormone receptors

BACKGROUND: Steroid hormone receptors constitute a special group of receptors having a wide range of efficiency and distribution in the body. Androgen and estrogen receptors, and their expression in the body, are linked with attributes such as reproduction control and sexual behaviour, but their relation with behavioural models, perception, memory and stress remain unclear to date. PURPOSE: In this project we aim to focus on monitoring the expressive influence of steroid hormone receptors on embryonic tissues and subsequently, expand our study to include the expression on adult tissues such as the CNS and to monitor the developmental aspects and relations pertaining to neurodegenerative disorders, such as Huntington's disease. MATERIAL AND METHODS: We shall rely on immuno-histochemistry, immuno-fluorescence and RT-PCR methods for detecting steroid hormone receptors and Huntingtin-associated protein 1 in the embryonic and adult tissue. CONCLUSION: Mapping the expression of steroid receptors during development represents an essential step in the quest for further studies and monitoring of the expression in adult tissues.     

Dual functions of the steroid hormone receptor coactivator 3 in modulating resistance to thyroid hormone

Mutations of the thyroid hormone receptor beta (TRbeta) gene cause resistance to thyroid hormone (RTH). RTH is characterized by increased serum thyroid hormone associated with nonsuppressible thyroid-stimulating hormone (TSH) and impaired growth. It is unclear how the actions of TRbeta mutants are modulated in vivo to affect the manifestation of RTH. Using a mouse model of RTH that harbors a knockin mutation of the TRbeta gene (TRbetaPV mouse), we investigated the effect of the steroid hormone receptor coactivator 3 (SRC-3) on RTH. In TRbetaPV mice deficient in SRC-3, dysfunction of the pituitary-thyroid axis and hypercholesterolemia was lessened, but growth impairment of RTH was worsened. The lessened dysfunction of the pituitary-thyroid axis was attributed to a significant decrease in growth of the thyroid and pituitary. Serum insulin-like growth factor 1 (IGF-1) was further reduced in TRbetaPV mice deficient in SRC-3. This effect led to reduced signaling of the IGF-1/phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway that is known to mediate cell growth and proliferation. Thus, SRC-3 modulates RTH by at least two mechanisms, one via its role as a receptor coregulator and the other via its growth regulatory role through the IGF-1/PI3K/AKT/mTOR signaling.     

Oligodeoxynucleotide base recognition by steroid hormone receptors

Oligodeoxynucleotides covalently linked to cellulose were used as probes of the DNA-binding domains of mouse steroid holoreceptors. With uterine cytosol estrogen receptor (E₂R) the relative binding order, in prior studies, was oligo(dG) > oligo(dT) ≧ oligo(dC) > > oligo(dA) > oligo(dI). The binding reactions were salt-sensitive with an optimal KCl concentration of 0.1–0.2 M. There was no enhancement of binding by activation, either temperature- or salt-induced. In the present study, using the oligomer ligands at a lower concentration, oligo(dT) binding was greater than that to oligo(dC). Quantitative differences in oligodeoxynucleotide binding were elicited by a number of inhibitors. These differences are again seen by exposure of E₂R to chaotropic salts such as SCN^?, ClO₄^? and NO₃^? as well as to putative modifiers of receptor amino acids, ie, iodoacetamide, 1,2 cyclohexanedione, and Rose Bengal. These results, and the quantitative differences following heat and purification, led to a designation of two types of subsites within the DNA-binding domain of uterine E₂R. These are stable G sites, which interact with oligo(dG); and labile N sites, which bind to oligo(dT), oligo(dC) and oligo(dA). Stimulation of binding to N sites and stabilization of the holoreceptor was effected by histones H2A and H2B. However, the differential response to incubation at 37°C was not altered by addition of H2B. Treatment of uterine E₂R by limited proteolysis also eliminated the stimulatory response to H2B. The above data, as well as prior studies, indicate that steroid holoreceptors can discriminate between the structural features of deoxynucleotide bases and this recognition process can be modulated by accessory proteins.