Synergistic activation of estrogen receptor with combinations of environmental chemicals.

Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.

SNX27 links DGKζ to the control of transcriptional and metabolic programs in T lymphocytes.

Sorting nexin 27 (SNX27) recycles PSD-95, Dlg1, ZO-1 (PDZ) domain-interacting membrane proteins and is essential to sustain adequate brain functions. Here we define a fundamental SNX27 function in T lymphocytes controlling antigen-induced transcriptional activation and metabolic reprogramming. SNX27 limits the activation of diacylglycerol (DAG)-based signals through its high affinity PDZ-interacting cargo DAG kinase ζ (DGKζ). SNX27 silencing in human T cells enhanced T cell receptor (TCR)-stimulated activator protein 1 (AP-1)- and nuclear factor κB (NF-κB)-mediated transcription. Transcription did not increase upon DGKζ silencing, suggesting that DGKζ function is dependent on SNX27. The enhanced transcriptional activation in SNX27-silenced cells contrasted with defective activation of the mammalian target of rapamycin (mTOR) pathway. The analysis of Snx27 -/- mice supported a role for SNX27 in the control of T cell growth. This study broadens our understanding of SNX27 as an integrator of lipid-based signals with the control of transcription and metabolic pathways.

Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs.

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis.

In vitro synergistic interaction of alligator and human estrogen receptors with combinations of environmental chemicals.

The effect of mixtures of environmental chemicals with hormonal activity has not been well studied. To investigate this phenomenon, the estrogen receptor (ER) from the American alligator (aER) or human (hER) was incubated with [3H]17beta-estradiol in the presence of selected environmental chemicals individually or in combination. The environmental chemicals included the insecticide chlordane, which has no estrogenic activity, and the pesticides dieldrin and toxaphene, which have very weak estrogenic activity. Chlordane, dieldrin, and toxaphene individually demonstrated no appreciable displacement of [3H]17beta-estradiol from aER and hER at the concentration tested. A combination of these chemicals inhibited the binding of [3H]17beta-estradiol by 20 to 40%. Alachlor, a chemical recently discovered to have weak estrogenic activity, also displaced [3H]17beta-estradiol more effectively in combination with dieldrin than alone. These results indicate that combinations of some environmental chemicals inhibit [3H]17beta-estradiol binding in a synergistic manner. This suggests that the ER may contain more than one site for binding environmental chemicals. The possibility that the ER binds multiple environmental chemicals adds another level of complexity to the interaction between the environment and the endocrine system.

Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum.

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution.

The estrogenic and antiestrogenic activities of phytochemicals with the human estrogen receptor expressed in yeast.

We have used the expression of the human estrogen receptor (hER) and two estrogen response elements linked to the lacZ gene in yeast (YES) to study the estrogenic and antiestrogenic activities of various phytochemicals. Coumestrol, alpha-zearalenol, or genistein could produce beta-galactosidase activity comparable to estradiol, but these required concentrations 100 to 1000-fold greater than estradiol. These compounds did not possess antiestrogenic activity. Narigenin, kaempferide, phloretin, biochanin A, flavone, or chrysin only partially induced beta-galactosidase activity in the YES at any concentration tested. When narigenin, kaempferide, or phloretin was given concurrently with estradiol, the estradiol-dependent beta-galactosidase activity was not inhibited by more than 50%. However, biochanin A, flavone, or chrysin could inhibit the activity of estradiol in a dose-response manner with IC50 values of 500 nM, 2 microM, and 10 microM, respectively. Combinations of biochanin A, chrysin, and flavone decreased estradiol-dependent beta-galactosidase activity in an additive fashion. Similar to the antiestrogens tamoxifen or ICI 182, 780, the antiestrogenic activity of these compounds with the exception of chrystin involved the disruption of hER dimerization, as demonstrated in the yeast two-hybrid system. Biochanin A, chrysin, or flavone were less effective in inhibiting the activity of an estrogenic polychlorinated biphenyl than they were inhibiting the activity of estradiol. Interestingly, this latter group of antiestrogenic phytocompounds did not inhibit the estrogenic activity of such phytochemicals as coumestrol or genistein. These results suggest that the antiestrogenic activity of biochanin A and flavone occurs by a mechanism similar to tamoxifen or ICI 182,780. Moreover, it seems that phytochemicals functioning as antiestrogens do not inhibit the activity of all estrogenic chemicals to the same extent. This suggests that conformational changes induced by different estrogens bound to the hER may regulate the antiestrogenic activity of a compound.

Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen.

We have used the yeast estrogen (YES) consisting of the human estrogen receptor and a reporter containing two estrogen response elements linked to the lacZ gene to evaluate the interaction between ovarian, phyto-, and synthetic estrogens with extracellular binding proteins. YES was incubated with charcoal-stripped human serum, human sex hormone-binding globulin, or human alpha-fetoprotein in the presence of concentrations of various estrogens that induced a 100% estrogenic response, as measured by beta-galactosidase activity. The activity of estradiol and coumestrol, a phytoestrogen, was reduced 75% with physiological levels of serum, sex hormone-binding globulin, or alpha-fetoprotein. The beta-galactosidase activity of genistein, another phytoestrogen, also decreased with extracellular proteins but to a lower extent than estradiol. In contrast, the activity of the synthetic estrogens diethylstilbestrol, kepone, and p,'p-DDD was only minimally reduced with extracellular proteins. These results indicate a potential fundamental difference in the interaction of estrogens from diverse sources with extracellular binding proteins. This suggests that the capacity for various estrogens to induce estrogen-associated responses is in part regulated by their affinity for extracellular bindings proteins.

Synergistic responses of steroidal estrogens in vitro (yeast) and in vivo (turtles).

Many environmental agents exert estrogenic activity. Previous studies from our laboratories demonstrated that certain combinations of environmental estrogens (i) reverse the sex of male turtle embryos in a synergistic manner (Bergeron et al., (1994) Environ. Hlth Perspect. 102, 780-782), and (ii) synergistically transactivate the human estrogen receptor (hER) in yeast and mammalian cells (Arnold et al., (1996) Science 272, 1489-1492). Because our findings with synthetic estrogens suggested that combinations of naturally-occurring steroidal estrogens might also produce synergistic activity of the ER, we used the same model systems to measure the activity of combinations of steroidal estrogens. The activity of combinations of estrone, estradiol-17beta or estradiol-17alpha in yeast strains expressing hER was synergistic at submaximal concentrations of both estrogenic compounds. However, synergy was not observed with mixtures of estrogens when the concentration of one of the estrogens alone was maximally active in yeast. Ligand-binding assays in yeast performed with various radiolabeled estrogens suggested that multiple estrogens may interact with the receptor. The estrogen-dependent process of sex-reversal of turtle embryos incubated at a male-producing temperature was used to determine whether steroidal estrogens also had synergistic activity in vivo. In this instance, a combination of estriol and estradiol-17beta was effective in reversing the gonadal sex of turtle embryos from males to females in a synergistic manner. Our results suggest that the synergy of some combinations of estrogens, synthetic or steroidal, may play a role in the estrogen-dependent process of sexual development in certain species.