Homology-Modeled Ligand-Binding Domains of Zebrafish Estrogen Receptors {alpha}, ß1, and ß2: From in Silico to in Vivo Studies of Estrogen Interactions in Danio rerio as a Model System

Aurora D. Costache, Phani Kumar Pullela, Purnachandar Kasha, Henry Tomasiewicz and Daniel S. Sem

Chemical Proteomics Facility, Department of Chemistry, Marquette University (A.D.C., P.K.P., P.K., D.S.S.),Milwaukee, Wisconsin 53201; and Marine and Freshwater Biomedical Sciences Center, University of Milwaukee (H.T.), Milwaukee, Wisconsin 53204

Address all correspondence and requests for reprints to: Dr. Daniel S. Sem, Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, Wisconsin 53201. E-mail: daniel.sem{at}marquette.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Homology models were constructed for the ligand-binding domains of zebrafish estrogen receptors (zfERs) {alpha}, ß1, and ß2. Estradiol-binding sites are nearly identical in zfERs and their human homologs, suggesting that zebrafish will serve as a good model system for studying human ER-binding drugs. Conversely, studies of endocrine disruptor effects on zebrafish will benefit from the wealth of data available on xenoestrogen interactions with human ERs. Compounds flagged by the Interagency Coordinating Committee on the Validation of Alternative Methods for endocrine disruptor screening were docked into our zfER homology models. Ideally, these in silico docking studies would be complemented with in vivo binding studies. To this end, fluorescently tagged estradiol was docked into zfER{alpha} and found to bind in the same manner as in human ER{alpha}, with fluorescein preferentially occupying a region between helices 11 and 12. Fluorescently tagged estradiol was synthesized and was found to localize along the path of primordial germ cell migration in the developing zebrafish embryo 3 d after fertilization, consistent with previous reports of 1) a role for estradiol in sex determination, and 2) the first appearance of ERs 2 d after fertilization. These data provide a foundation for future in silico and in vivo binding studies of estrogen agonists and antagonists with zebrafish ERs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ZEBRAFISH ARE INCREASINGLY being used as a model system to study human disease, because they have close homologs of many human proteins, including kinases (1), most proteins of mammalian apoptotic pathways (2), estrogen receptors (ERs) (3, 4), and aromatase (5). Zebrafish have been used to screen for the biological effects of drugs (1, 6, 7) as well as their undesired toxic side effects (8, 9, 10, 11). Zebrafish are also used to study the effects of pollutants, especially endocrine disruptors that are thought to work by binding to ERs (12). It is known that some pollutants disturb normal sexual development and reproduction (13) and can affect gene expression via regulatory proteins such as the ER. Indeed, interactions with the ER (along with androgen receptor) are thought to be the primary triggering event leading to the adverse effects of endocrine disruptor pollutants, which are being increasingly monitored by the Environmental Protection Agency (14). Some of these pollutants are recognized as estrogen molecules by fish and human cells (15, 16, 17) and can bind to ERs found in ovaries, breast, liver, brain, bone, and other target tissues. They can activate or inhibit the receptor, which initiates a series of changes that can affect sexual development. To address this endocrine disruptor problem, the National Institute of Environmental and Health Sciences has a mandate to identify and validate assays for estrogen and androgen receptor-based activities of compounds. These assays should be "more predictive of human and ecological effects," to identify endocrine disruptors (18). To this end, an interagency committee [Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)] has begun work comparing assay methods and results for an initial set of 78 recommended test substances for which significant experimental data are already available (18). Our in silico docking studies with zebrafish ERs were therefore performed with these recommended test substances, because they are already well characterized in terms of ER binding and endocrine disruptor potential. Because some of these compounds were either simple salts or polycyclic hydrocarbons with no functional groups, docking was performed on only 70 of the 78 compounds (Table 1GoGo).


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Table 1. zfER Docking Scores

 

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Table 1A. Continued

 
The ERs are ligand-activated transcription factors that participate in the regulation of many processes, including development and reproduction. Agonists cause ER to adopt an activated state, whereas antagonists inhibit estrogen binding and cause the receptor to adopt an inactive state (19). Interestingly, the agonist or antagonist behavior of an estrogen-like molecule can vary depending on the tissue, suggesting that the in vivo environment plays a role in protein-ligand interactions. Tamoxifen (20) is a breast cancer therapeutic that in bone, uterus, and the cardiovascular system plays an agonist role. In contrast, raloxifene (19) acts as an antagonist in breast and uterus, but functions as an agonist in bone and the cardiovascular system. Drugs such as these, which have variable agonist/antagonist activity depending on the tissue, are called selective ER modulators. The existence of such tissue-dependant behavior of drugs and pollutants needs to be better understood at the molecular level, and in vivo binding assays in model organisms such as zebrafish may 1) provide such mechanistic understanding, and 2) allow the refinement of tools for studying ER-ligand binding interactions in vivo.

In this paper we demonstrate the similarity of the ligand-binding domains of human and zebrafish ERs. Crystal structures for numerous human ER{alpha} and ERß are available (19, 20, 21, 22), but no structures are available for zebrafish ERs (3, 4). In this study we present homology models of zebrafish ERs, developed using human ER crystal structures as templates. Although sequence identity among these structures was found to be 50–70%, the identity of binding site residues is even higher. This similarity suggests that zebrafish are an excellent human model system for in vivo screening of interactions with ERs. Such studies are especially attractive in zebrafish, because the developing embryo is transparent, permitting the detection of fluorescent probes in vivo. This property has been widely used to monitor the production of green fluorescent proteins for applications that include screening endocrine disruptors (12) and monitoring the localization of primordial germ cells during sexual development (23, 24). Our zebrafish ER (zfER) models are used to show the utility of a fluorescently tagged estradiol (F-E2) for in vivo studies of protein-ligand interactions. Initial data suggest that such in vivo fluorescence assays could be used to probe ER interactions that involve migration of primordial germ cells during sexual development. Potential endocrine disruptors to be tested in this assay can be identified based on in silico docking studies using our four zfER models.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Homology Modeling
Alignments of zfER isoforms with their human homologs are summarized for ER{alpha} (Fig. 1AGo) and ERß1/ERß2 (Fig. 1BGo), with secondary structures indicated above each alignment. Secondary structures are from the homology models, as calculated in the next section. Sequence identities between zebrafish ERs and their human homologs are always greater than 50%. Specifically, zebrafish ER{alpha} is 59% identical with its human homolog, whereas zebrafish ERß1 and ERß2 are 70% and 68% identical, respectively, with human ERß. Noteworthy is that zfERß1 and zfERß2 show the highest identity to the human ERß, whereas the zfER{alpha} shows comparable identity to human ER{alpha} and ERß, suggesting that zfER{alpha} is to some extent a hybrid of the two isoforms or at least evolutionarily more distant from its human homolog.



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Fig. 1. Sequence Alignments of ER Ligand-Binding Domains

Alignments represent the regions that were homology modeled. Sequence identities are indicated with gray boxes, and binding site residues are indicated in bold. A, Sequence alignment of zebrafish and human ER{alpha}. The secondary structure of the zebrafish ER{alpha} homology model is represented above the alignment as cylinders for {alpha}-helices and bars for extended regions. Numbers are provided for starting and ending residues of {alpha}-helices, and binding site residues are indicated in bold. B, Sequence alignment of the two zebrafish ERß isoforms with human ERß, with secondary structures represented as described in A for the homology models. Sequence alignments and percent identities were calculated using MOE (43 44 ) software (Chemical Computing Group) and BLAST (BLAST: http://www.ncbi.nlm.nih.gov/BLAST/).

 
Homology models were constructed for all zfER ligand-binding domains using MODELLER (25, 26, 27); zfER{alpha} was modeled using both ER{alpha} agonist (Protein Data Bank ID code 1ERE) and antagonist (1ERR) structures as templates to generate corresponding agonist and antagonist zebrafish ER structures. Likewise, zfERß1 and zfERß2 were modeled using human ERß (1L2J) as a template. Homology models constructed based on templates that have greater than 50% sequence identity to the target sequence have previously been compared with crystal structures of about 3 Å resolution (25), which provides an approximate measure of the resolution of our models.

Analysis of zfER{alpha}, zfERß1, and zfERß2 Homology-Modeled Structures
The homology models of the ligand-binding domains of zfERs were overlaid with the human ER homologs (Fig. 2Go). For the overlay of all four zebrafish homology models, the backbone root mean square deviation (RMSD) for C{alpha} atoms 1–187 (excluding helices 11 and 12) was 2.47 Å. The overlay of the two zfER{alpha} models on the five human ER{alpha} crystal structures gave an RMSD of 0.92 Å, whereas the overlay of the zfERß1 and zfERß2 models on the three human ERß crystal structures gave an RMSD of 1.37 Å. The movement of helix 12 is highlighted with arrows to indicate the differences between the structures when an agonist vs. an antagonist is bound (Fig. 2Go, A and B). The human ER{alpha} structures, which are represented in dark gray in Fig. 2AGo, are bound to the agonists: estradiol, diethylstilbestrol (DES), or (R,R)-5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (THC). They have helix 12 packed tightly over the estradiol-binding site, whereas the antagonists raloxifene and 4-hydroxytamoxifen displace helix 12 so that it occupies the coactivator site. The homology-modeled structures of zfERß1 and zfERß2, which are represented as light gray and white in Fig. 2CGo, are overlaid on two human ERß crystal structures complexed with either THC or genistein. There is a nearly perfect overlay in all regions, including helix 12. Helix 12 of ERß1 and ERß2 is orientated similarly to helix 12 of ER{alpha} in the antagonist conformation, and there is only modest rearrangement of helix 11. In summary, the overall folds of zfER{alpha}, zfERß1, and zfERß2 are very close to their human homologs.



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Fig. 2. Comparison of Homology-Modeled zfER{alpha}, zfERß1, and zfERß2 with Their Human Homologs

A and B, Homology modeled zfER{alpha} (white, agonist model; gray, antagonist model) overlaid on human ER{alpha} bound to various ligands. Human structures of agonist complexes are identified with light gray arrows: 17ß-estradiol (1ERE), DES/glucocorticoid receptor-interacting protein (3ERD), and THC/glucocorticoid receptor-interacting protein (1L2I). Human structures of antagonist complexes are identified with dark gray arrows: raloxifene (1ERR) and 4-hydroxytamoxifen (3ERT). All agonist structures (three human and one zebrafish) overlay so closely that individual structures cannot be distinguished, as is the case for the antagonist structures (two human and one zebrafish). A, Full structures of ligand-binding domains, with agonist and antagonist orientations of helix 12 indicated with light and dark gray arrows, respectively. B, Same as A, but with only helices 11 and 12 shown along with bound ligands. Antagonists can be seen to extend into the region where helix 12 resides in the agonist structures, but the protein adopts a conformation with helix 12 now rotated approximately 90° in the antagonist relative to agonist complexes. The perspective is looking down helix 11 in the N to C direction. C and D, Homology modeled zfERß1 (white) and zfERß2 (gray) overlaid on human ERß (dark gray) bound to either THC (1L2J) or genistein (1QKM). As before, the overlay is shown for the full ligand-binding domain (C) and a view of the helix 11/12 region (D).

 
The main regions of sequence variability between the human and zebrafish structures are between helices 2 and 3 as well as between helices 9 and 10. This variability is especially pronounced for the zebrafish ß1 and ß2 isoforms, which show small insertions relative to human ERß. The region between helices 9 and 10 is in a large loop that is distal from the estradiol-binding site. The region between helices 2 and 3 is in an extended region of surface structure that is also far from the binding site and helix 12. Therefore, both of these variable regions are unlikely to play any direct role in binding hormone. The insertion between helices 2 and 3 is somewhat basic (Lys-Lys), whereas that between helices 9 and 10 is characterized by the presence of a cysteine residue as well as a Ser-Ser pair. The function of these variable regions is not known. Another notable amino acid substitution is the human Asp321->zebrafish Gly14 change in ER{alpha}, but these residues are also on the surface and therefore are unlikely to play any direct role in binding. The human Cys381/Ala382->zebrafish Ser74/Ser75 substitution in ER{alpha} is in the vicinity of the pocket occupied by helix 12 and so could affect coactivator binding.

More important than the overall fold and the variable surface loops is the composition of the estradiol-binding pocket. Binding site residues in zebrafish and human ERs are identified in Fig. 1Go. Assignment as an active site residue is based on previous analyses of crystal structures of human ER{alpha} (19) and ERß (20) bound to either agonist or antagonist ligands. In Fig. 3Go, A and B, are shown binding site amino acid overlays for zebrafish models and human templates, with ligands present. In Fig. 3AGo are overlaid the amino acids from the binding site of the zfER{alpha} agonist model and the human crystal structure for ER{alpha}. The red/blue residues represent the Leu349 in human and the Met42 in zebrafish. In Fig. 3BGo are overlaid the amino acids from the binding sites of the zebrafish ERß2 model and the human crystal structure of ERß bound to genistein (1QKM). Analysis of the binding site amino acid residues identified in Fig. 1Go and shown in Fig. 3AGo indicates that the only difference between the binding sites of human and zebrafish ER{alpha} is the replacement of Leu349 with Met42 (zebrafish numbering is only for the ligand-binding domain), which is located in helix 3. Although this is a relatively conservative substitution, it could affect binding of THC, because comparison with the crystal structure of THC-ER{alpha} (1L2I) (21) indicates that this residue is in contact with the THC ligand. However, for most ER{alpha} ligands it is expected that they will bind in the same manner to both zebrafish and human ER{alpha}. Human ERß-binding site amino acids were also compared with the two zfERß models (Fig. 3BGo), and five substitutions were found: Met336 to Leu77, Ile356 to Leu114, and Val485 to Ala227 in the ERß2 model, and Met473 to Ile215 and Val487 to Leu229 in the ERß1 model. Although these substitutions are somewhat conservative, they might have some effect on the binding affinity for ERß ligands because there are a number of changes that collectively alter binding site shape. All models have the same His524 residue (numbering for human structure), which interacts with the 17ß-hydroxyl of the D ring of estradiol through a hydrogen bond (28). As with the analysis of overall fold, the estradiol-binding sites in zfER{alpha}, zfERß1, and zfERß2 are very similar to their human homologs, and most differences are not readily distinguishable within the resolution of our models, but the binding sites of zfERß1/zfERß2 are more divergent from their human homologs compared with the ER{alpha}s.



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Fig. 3. Comparison of Active Sites for Zebrafish and Human ER Isoforms

A, Overlay of all active site residues for human ER{alpha} agonist (blue or green) and zfER{alpha} agonist (red or purple) structures, with side chains identified for all binding site residues specified in Fig. 1Go. Residue numbers are for the zebrafish sequence. B, Overlay of all active site residues for human ERß (blue or green) and zfERß1 (red or purple). C, Comparison of estradiol docked into zfER{alpha} (agonist conformation) with the crystal structure of the estradiol-ER{alpha} binary complex (1ERE). Distances to key binding site residues (amino acid numbering for the human structure) are provided in angstroms. The human structure is purple, and the zebrafish structure is light blue. Docking into the zebrafish structure was performed with MOEDOCK, and both were minimized with Amber (45 ).

 
In Silico Docking of Estradiol into zfER{alpha}
The zebrafish ER{alpha} model was used for docking calculations with estradiol. As shown in Fig. 3CGo, estradiol docked into our model as expected. Our model with docked estradiol aligned with the crystal structure of human ER{alpha} (1ERE) to give an RMSD (C{alpha}; residues 1–187) of 1.7 Å. The lowest energy structure (–21 kcal/mol) was overlaid with estradiol in the human ER{alpha} structure. The amino acids that make direct hydrogen bonds are shown, with distances indicated by dashed lines connecting the proximal atoms. This docking study shows that our zfER{alpha} model is very similar to human ER{alpha}, and that the estradiol docked in the zfER{alpha} model binds in the same manner as estradiol in the human ER{alpha} crystal structure. Therefore, the specificity for estradiol analogs, such as endocrine disruptors, xenoestrogens, and estrogen antagonists used as breast cancer therapeutics, is likely to be very similar, if not identical, between zfER{alpha} and human ER{alpha}.

In Silico Docking of Potential Endocrine Disruptors into zfERs
The test compounds (70 of 78) chosen by the ICCVAM for development and validation of endocrine disruptor assays were docked into the zfER structures using Autodock (29). Docking scores in rank order from lowest to highest (highest affinity) are given in Table 1GoGo, and previously reported assay results (relative binding affinity) from experimental binding studies with human ER{alpha} are provided as well for reference. Docked structures (protein not shown) are given for several well-known ER ligands (Fig. 4Go), and these are oriented roughly as expected, providing some validation for the docking calculations. Although rank ordering of relative binding affinity based on Autodock energies was poorly correlated with known binding affinities for human ERs, somewhat better correlation was found using the X-score energy scoring function. This is consistent with previous reports that it is best to separate the docking and scoring process, and that X-score’s scoring function is more predictive than Autodock’s (30). The best predictability was achieved using the average of the three scores calculated by X-score and also by averaging these scores for binding to all four zfER models. With this strategy, it was possible to predict nine of the 11 compounds with relative affinity at least 2% that of 17ß-estradiol, using an averaged X-score value of at least 6.5 [–log(Kd)]. Although there were a number of false positives (nonbinders) with X-score values greater than 6.5, and two strong binders were missed (false negatives), these calculations at least serve to prioritize compounds to be tested as endocrine disruptors. Nearly as good a correlation was found using average docking energies from only the ER{alpha} structures (last column), although DES was scored too weakly relative to other compounds, and would therefore have been missed. We suspect that the somewhat better predictability achieved by including the ERß1 and ERß2 structures is simply an artifact of the fact that we are docking into an artificially constrained binding site by using rigid zfER{alpha} structures. Therefore, future docking studies will consider ER binding site flexibility. Furthermore, docking predictions (Table 1GoGo) will be tested using in vitro binding assays with zfERs (when available), and with in vivo binding studies using a displacement assay variant of the in vivo assay described below. The ultimate goal of these studies is to create a large database of endocrine disruptors docked into our zfER models along with binding affinities and to use these data to construct three-dimensional quantitative structure-activity relationship models for predicting likely endocrine disruptor activity, thereby permitting the flagging of compounds for follow-up in vitro and in vivo assays (31).



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Fig. 4. In Silico Dockings into zfER Models Compared with Human ER Structures

A, Overlay of known ER ligands, as docked with Autodock (29 ). The lowest energy docking poses are shown, along with key binding site residues Arg394, Glu353, and His524 from human ER{alpha}, which are involved in key interactions with hydroxyl groups on opposite ends of estradiol. Oxygen atoms are rendered as balls (structures are colored: green, 17ß-ethinyl estradiol; yellow, 4-hydroxytamoxifen; red, DES; orange, genistein; blue, tamoxifen) and by atom type (17{alpha}-estradiol). B, DES docked into zfER{alpha} (purple) and overlaid on the crystal structure (3ERD) (20 ) of the human ER{alpha}/DES complex (green), with atoms only shown for the docked DES ligand. C, Genistein docked into zfERß1 (purple) and overlaid on the crystal structure (1QKM) (22 ) of the human ERß/genistein complex (green), with atoms only shown for the docked genistein ligand.

 
In Silico Docking of F-E2 into zfER{alpha}: Implications for Fluorescence Studies
We are interested in developing the use of a fluorescently tagged estradiol (F-E2) for screening compounds that bind to zfER, both in vitro and in vivo. Such binding studies would offer an experimental follow-up validation to in silico prescreening of larger numbers of compounds with suspected affinity for zfERs (see above). Because it has previously been shown that estradiol fluorescently tagged at the 17ß position can bind to human ERs (32, 33), we decided to use the same strategy for zfERs, as long as it could be established that binding to zfERs would have similar structural requirements as those for the human ERs. Unfortunately, there are no crystal structures of any zfER, and although there are many structures of human ERs, there are no structures or even models with F-E2 bound. We therefore manually docked F-E2 (Fig. 5AGo) into our zfER homology models. As expected, based on the high structural relatedness of human and zebrafish ERs, the F-E2 fits easily into the modeled ER{alpha} structure (Fig. 5BGo). The fluorescein fits best on the side of helix 11 closest to helix 12, although it is also possible to position it on the other side of helix 11, but this requires a small global rearrangement, which is less likely to occur. Noteworthy is that the fluorescein binds in such a way that a coactivator peptide, modeled in from the corresponding structure of human ER{alpha} with a transcriptional intermediary factor 2 (TIF2) peptide bound (32), can still bind properly. Thus, the modeling predicts that F-E2 will bind as easily to zfER{alpha} as to human ER{alpha}. The fluorescein group is located in a cleft on the surface of the protein (Fig. 5BGo), but is still close enough to the protein that motion will be restricted and therefore lead to a large fluorescence polarization signal, which is useful for in vitro screening of endocrine disruptors (33). Likewise, the ß1 and ß2 zfER isoforms will also tolerate the fluorescent tag (data not shown) with some rearrangement of loop regions. Binding of F-E2 is therefore likely to occur for zfERß1 and zfERß2 also. This would be consistent with a previous report that F-E2 can bind to human ERß (34).



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Fig. 5. Fluorescence Probe for in Vivo Estradiol Binding

A, Chemical structure for F-E2 that was injected into zebrafish embryos. B, Two possible binding modes for F-E2, with the fluorescein on the side of helix 11 that is proximal (energy, –4401 kcal/mol; protein is light gray) or distal (energy, –4705 kcal/mol; protein is dark gray) to helix 12. These two structures have been overlaid on the original structure of the human estradiol-ER{alpha} binary complex, shown in medium gray (1ERE). To orient relative to the coactivator site, the TIF2 coactivator peptide was modeled into the 1ERE structure by superimposing the structure of the TIF2-estradiol-ER{alpha} ternary complex (1GWR), then deleting the estradiol and the ER{alpha} protein.

 
Fluorescence Studies of F-E2 Binding Interactions in Zebrafish: Toward an in Vivo Binding Assay for Xenoestrogens
We have synthesized the F-E2 molecule shown in Fig. 5AGo. This compound was injected into embryos shortly after fertilization, and fluorescence localization was monitored daily. Relative to a control injection with fluorescein, no significant localization was found until 3 d postfertilization (dpf), at which point significant fluorescence was observed in regions that appear to be on the path of migration of the primordial germ cells (PGCs), which serve as precursors to the gonads (23, 24). Such localization is of interest because steroids such as estrogen are known to play a role in sex determination (35, 36, 37, 38). That our 17{alpha}-substituted F-E2 should bind well in vivo during sexual development is not unexpected, because previous studies with a 17{alpha}-substituted ethinyl estradiol had shown this analog to affect sexual development in zebrafish (39, 40). In terms of where fluorescence was localized, Tanaka et al. (41) had observed localization of a green fluorescent protein PGC marker in transgenic Oryzias latipes (a teleost related to Danio rerio) in the ventrolateral region of the posterior intestine at stage 25, but they noted that although there was general posterior migration of PGCs, there was fluorescent signal that remained ectopically in anterior positions later in development. Their studies were directed toward the development of an assay for pollutant effects on the development of gonads in teleosts. Weidinger et al. (23) measured the migration of PGCs in zebrafish, which they characterized as movement toward an attractant coming from both intermediate and final target locations. They observed PGCs in anterior, medial, and posterior positions throughout the course of gonadal development. The intermediate target where PGCs localized gave rise to the prenephros, whereas further migration of PGCs to a more posterior position resulted in gonad development. We believe that the localization of F-E2 observed in Fig. 6BGo reflects the path of migration of the PGCs. Such localization is also correlated in time with the presence of an estrogen-binding protein (most likely ER{alpha}). Our observation of fluorescence signal at 3 dpf is also consistent with a previous observation (42) that ERs, especially ER{alpha}, first appear in zebrafish at 2 dpf. The localization observed in Fig. 6BGo is consistent with the observation by Weinger et al. (23) that PGCs do not reach their final target by 1 dpf, and that by Tanaka et al. (41) that PGCs can remain behind ectopically during later stages of development. Thus, although our in vivo F-E2 binding studies are preliminary, they do suggest a possible in vivo competition assay for xenoestrogens at the level of protein-ligand interactions to identify compounds with a potential role in sexual development in zebrafish. The optimal screening time may be at 3 dpf, but will most likely require the use of another fluorescent label, such as rhodamine, because we observed some background localization of fluorescein alone (Fig. 6CGo) in a more dorsal location relative to the F-E2 signal (Fig. 6BGo). Nevertheless, it should be noted that the relative concentration of F-E2 (Fig. 6BGo) is probably higher than that of fluorescein (Fig. 6CGo), because the intrinsic fluorescence of F-E2 is at least 8-fold less than that of fluorescein due to internal quenching of F-E2 (data not shown).



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Fig. 6. F-E2 Binding in Vivo: Localization of Functional ER during Development

Fluorescence images (489-nm excitation filter) of zebrafish at 3 dpf, after being injected on the morning of fertilization with either 10 µM F-E2 (B) or 10 µM fluorescein (C), compared with uninjected fish (A). Each image is a representative fish, and a major fluorescence signal is indicated with arrows. Significant localization of fluorescent signal is noted with arrows.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
All chemical and biochemical reagents were obtained from Sigma-Aldrich Corp. (St. Louis, MO), except for fluorescein isothiocyanate, which was purchased from Molecular Probes (Eugene, OR).

Construction of Homology Models
Alignments between zebrafish and human ERs for homology modeling are shown in Fig. 1Go. For modeling involving human ER{alpha} structures [protein data bank files 1ERE (19), 1ERR (19), 3ERD (20), 3ERT (20), and 1L2I (21)], the corresponding protein sequence accession number was P03372. For all modeling involving human ERß structures [protein data bank files 1L2J (21) and 1QKM (22)], the corresponding protein accession number was Q92731. Zebrafish sequences used were NP_694491 (42) (zfER{alpha}), CAC93848 (4) (zfERß1), and AAN60793 (3) (zfERß2).

The alignment of structures was performed using the MOE (43, 44) software package, based only on the first 187 amino acids of the models, to leave helices 11 and 12 free to adopt any appropriate orientation. Homology-modeled structures were constructed using MODELLER (25, 26, 27) software, followed by minimization under the Amber force field (45) using a distance-dependent dielectric constant.

Quality Assessment of the Models
A Prosa (46) analysis was performed to create energy graphs that provide diagnostic indicators of poorly calculated/folded regions of tertiary structure (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). No regions had positive energies, and z-scores varied from –6.8 to –8.3 (47). A PROCHECK (48, 49) (PROCHECK: http://biotech.ebi.ac.uk) analysis of the structures provided scores of overall quality of the structures, as well as other structural indicators. Ramachandran plots, calculated using RAMPAGE (RAMPAGE: http://raven.bioc.cam.ac.uk/rampage.php), indicated that 90% of amino acids were in the most favored regions, and 3% were in outlier regions (see supplemental data).

Docking Studies
Docking calculations for estradiol were made with our zebrafish ER{alpha} agonist model and compared with estradiol bound in the human ER{alpha} crystal structure. Energies were calculated with the Amber force field in the MOE software package (Chemical Computing Group). The 17ß-estradiol structure was first minimized using the MMFF94 force field (50, 51). The docking box was 48 x 48 x 48 Å surrounding the binding site of estradiol. Then 25 docking runs were performed using random starting orientations. Optimization was achieved with simulated annealing, using an initial temperature of 1000 K and six cycles per run. For docking the 70 compounds from the ICCVAM, all four zebrafish ER models were used. Compounds can be viewed at www.marquette.edu/cpfm, prepared using Pipeline Pilot software (SciTegic, Inc., San Diego, CA). Because most potential endocrine disruptors will have multiple rotatable bonds (unlike estradiol), flexible ligand docking was achieved using Autodock (29). Kollman charges were used along with standard solvation parameters. The docking grid box was 60 x 60 x 60 Å, with 0.508 Å between grid points, and was centered in the middle of the binding site. Optimization was with a genetic algorithm, and energies for the best of 10 docked structures are shown in Table 1GoGo, rank ordered based on average X-score (52) energies for all four zfER complexes. Average energies considering only the ER{alpha} complexes are also given (last column) for comparison.

Synthesis of F-E2
Synthesis of the 17-substituted estrone derivative (E2-NH2), containing a 4-amino-butyne substituent, was by a modification of the procedure reported by Ohno et al. (53). Although the synthesis was largely as described, introduction of protective nitrogen functionality and subsequent steps were modified for synthetic convenience (see supplemental data). The target compound (F-E2) was obtained by reaction of the amine of E2-NH2 with fluorescein isothiocyanate in dry dimethyl formamide containing 5% pyridine.

Zebrafish Injections
Zebrafish were maintained and embryos were raised at the National Institute of Environmental and Health Sciences Marine and Freshwater Biomedical Sciences Center using standard procedures (54) and in accord with accepted procedures of humane animal care. Embryos were microinjected using an Eppendorf Transjector (Eppendorf AG, Hamburg, Germany) before the 16-cell stage and were maintained at 28 C. Injections were of either 10 µM fluorescein or 10 µM F-E2 in 1% methanol in Danieau’s buffer [58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, and 5.0 mM HEPES (pH 7.6)]. The injection volume was approximately 10% that of the embryo. Only embryos containing fluorescence were retained, and they were monitored daily. Fluorescence images were obtained using an Olympus SZX12 upright dissecting stereomicroscope (New Hyde Park, NY) equipped with a filter for selective excitation of fluorescein. Exposure time was kept constant for all images, which were processed using Image Pro software (Visiopharm, Horsholm, Denmark).


    ACKNOWLEDGMENTS
 
We are grateful to the National Institute of Environmental and Health Sciences Marine and Freshwater Biomedical Sciences Center for the use of their facility and equipment.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grant ES-04184.

First Published Online August 4, 2005

Abbreviations: DES, Diethylstilbestrol; dpf, days postfertilization; ER, estrogen receptor; F-E2, fluorescently tagged estradiol; PGC, primordial germ cell; RMSD, root mean square deviation; THC, (R,R)-5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol; TIF2, transcriptional intermediary factor 2; zfER, zebrafish estrogen receptor.

Received for publication October 25, 2004. Accepted for publication July 25, 2005.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Parng C, Seng WL, Semino, C, McGrath P 2002 Zebrafish: a preclinical model for drug screening. Assay Drug Dev Technol 1:41–48[CrossRef][Medline]
  2. Inohara N, Nunez G 2000 Genes with homology to mammalian apoptosis regulators identified in zebrafish. Cell Death Differ 7:509–510[CrossRef][Medline]
  3. Lassiter CS, Kelley B, Linney E 2002 Genomic structure and embryonic expression of estrogen receptor ßa (ERßa) in zebrafish (Danio rerio). Gene 299:141–151[CrossRef][Medline]
  4. Menuet A, Pellegrini E, Anglade I, Blaise O, Laudet V, Kah O, Pakdel F 2002 Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions. Biol Reprod 66:1881–1892[Abstract/Free Full Text]
  5. Kishida M, Callard GV 2001 Distinct cytochrome P450 aromatase isoforms in zebrafish (Danio rerio) brain and ovary are differentially programmed and estrogen regulated during early development. Endocrinology 142:740–750[Abstract/Free Full Text]
  6. Langheinrich U 2003 Zebrafish: a new model on the pharmaceutical catwalk. Bioessays 25:904–912[CrossRef][Medline]
  7. Beckwith LG, Moore JL, Tsao-Wu GS, Harshbarger JC, Cheng KC 2000 Ethylnitrosourea induces neoplasia in zebrafish (Danio rerio). Lab Invest 80:379–385[Medline]
  8. Zhang C, Willett C, Fremgen T 2003 Zebrafish: an animal model for toxicological studies. Curr Protoc Toxicol 17(Suppl):1–18
  9. Milan DJ, Peterson TA, Ruskin JN, Peterson RT, MacRae CA 2003 Drugs that induce repolarization abnormalities cause bradycardia in zebrafish. Circulation 107:1355–1358[Abstract/Free Full Text]
  10. Stern HM, Zon LI 2003 Cancer genetics and drug discovery in the zebrafish. Nat Rev Cancer 3:533–539[CrossRef][Medline]
  11. Zambrowicz BP, Turner CA, Sands AT 2003 Predicting drug efficacy: knockouts model pipeline drugs of the pharmaceutical industry. Curr Opin Pharmacol 3:563–570[CrossRef][Medline]
  12. Carvan MJ, Dalton TP, Stuart GW, Nebert DW 2000 Transgenic zebrafish as sentinels for aquatic pollution. Ann NY Acad Sci. 919:133–147
  13. Berberich S 2002 Sex genes of fish disrupted by common household products. Univ MD Biotechnol Inst Natl Geographic News
  14. Fenner-Crisp PA 1997 Endocrine disruptor risk characterization: an EPA perspective. Regul Toxicol Pharmacol 26:70–73[CrossRef]
  15. U.S. Environmental Protection Agency 1997 Special report on environmental endocrine disruption: an effects assessment and analysis. EPA/630/R-96/012: 5
  16. Golub, Man S, Donald JM, Reyes JA 1991 Reproductive toxicity of commercial PCB mixtures: LOAELs and NOAELs from animal studies. Environ Health Perspect 94:245–253[Medline]
  17. Carnevali C, Galassi S, Bonasoro, Patruno M, Thorndyke MC 2001 Regenerative response and endocrine disrupters in crinoid echinoderms: arm regeneration in Antedon mediterranea after experimental exposure to polychlorinated biphenyls. J Exp Biol 204:835–842[Abstract/Free Full Text]
  18. 2003 ICCVAM evaluation of in vitro test methods for detecting potential endocrine disruptors: estrogen receptor and androgen receptor binding and transcriptional activation assays. Bethesda: National Institutes of Health; Publication 03-4503
  19. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson J-A, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  20. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[CrossRef][Medline]
  21. Shiau AK, Barstad D, Radek JT, Meyers MJ, Nettles KW, Katzenellenbogen BS, Katzenellenbogen JA, Agard DA, Greene GL 2002 Structural characterization of a subtype-selective ligand reveals a novel mode of estrogen receptor antagonism. Nat Struct Biol 9:359–365[Medline]
  22. Pike ACW, Brzozowski AM, Hubbard RE, Bonn T, Thorsell A-G, Engstrom O, Ljunggren J, Gustafsson J-A, Carlquist M 1999 Structure of the ligand-binding domain of oestrogen receptor ß in the presence of a partial agonist and a full antagonist. EMBO J 18:4608–4618[Abstract/Free Full Text]
  23. Weidinger G, Wolke U, Koprunner M, Thisse C, Thisse B, Raz E 2002 Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target. Development 129:25–36[Abstract/Free Full Text]
  24. Hsiao C-D, Tsai H-J 2003 Transgenic zebrafish with fluorescent germ cell: a useful tool to visualize germ cell proliferation and juvenile hermaphroditism in vivo. Dev Biol 262: 313–323
  25. Marti-Renom MA, Stuart AC, Fiser A, Sanchez R, Melo F, Sali A 2000 Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 29:291–325[CrossRef][Medline]
  26. Sali A, Blundell TL 1993 Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815[CrossRef][Medline]
  27. Fiser A, Do RK, Sali A 2000 Modeling of loops in protein structures, Protein Sci 9:1753–1773
  28. Aliau S, Mattras H, Richard E, Bonnafous JC, Borgna JL 2002 Differential interactions of estrogens and antiestrogens at the 17ß-hydroxyl or counterpart hydroxyl with histidine 524 of the human estrogen receptor {alpha}. Biochemistry 41:7979–7988[CrossRef][Medline]
  29. Goodsell DS, Morris GM, Olson AJ 1996 Automated docking of flexible ligands: applications of AutoDock. J Mol Recognit 9:1–5[CrossRef][Medline]
  30. Wang R, Lu Y, Wang S 2003 Comparative evaluation of 11 scoring functions for molecular docking. J Med Chem 46:2287–2303[CrossRef][Medline]
  31. Suzuki S, Ohno K, Santa T, Imai K 2003 Study on interactions of endocrine disruptors with estrogen receptor-ß using fluorescence polarization. Anal Sci 19:1103–1108[CrossRef][Medline]
  32. Warnmark A, Treuter E, Gustafsson JA, Hubbard RE, Brzozowski AM, Pike AC 2002 Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivator binding site of estrogen receptor {alpha}. J Biol Chem 277:21862–21868[Abstract/Free Full Text]
  33. Ohno K, Suzuki S, Fukushima T, Maeda M, Santa T, Imai K 2003 Study on interactions of endocrine disruptors with estrogen receptor using fluorescence polarization. Analyst 128:1091–1096[CrossRef][Medline]
  34. Waller CL, Oprea TI, Chae K, Park HK, Korach KS, Laws SC, Wiese TE, Kelce WR, Gray LE 1996 Ligand-based identification of environmental estrogens. Chem Res Toxicol 9:1240–1248[CrossRef][Medline]
  35. Baroiller JF, Guiguen Y, Fostier A 1999 Endocrine and environmental aspects of sex differentiation in fish. Cell Mol Life Sci 55:910–931[CrossRef]
  36. Francis RC 1992 Sexual lability in teleosts: developmental factors. Q Rev Biol 67:1–18[CrossRef]
  37. Pandian TJ, Koteeswaran R 1999 Lability of sex differentiation in fish. Curr Sci 76:580–583
  38. Yamazaki F 1983 Sex control and manipulation in fish. 33:329–354
  39. Islinger M, Willimski D, Volkl A, Braunbeck T 2003 Effects of 17{alpha}-ethinylestradiol on the expression of three estrogen-responsive genes and cellular ultrastructure of liver and testes in male zebrafish. Aquat Toxicol 62:85–103[CrossRef][Medline]
  40. Orn S, Holbech H, Madsen TH, Norrgren L, Petersen GI 2003 Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone. Aquat Toxicol 65:397–411[Medline]
  41. Tanaka M, Kinoshita M, Kobayashi D, Nagahama Y 2001 Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cells in a live vertebrate. Proc Natl Acad Sci USA 98:2544–2549[Abstract/Free Full Text]
  42. Bardet PL, Horard B, Robinson-Rechavi M, Laudet V, Vanacker JM 2002 Characterization of oestrogen receptors in zebrafish (Danio rerio). J Mol Endocrinol 28: 153–163
  43. Berger MP, Munson PJ 1991 A novel randomized iterative strategy for aligning multiple protein sequences. Comput Appl Biosci 7:479–484[Abstract]
  44. Hirosawa M, Totoki Y, Hoshida M, Ishikawa M 1995 Comprehensive study on interactive algorithms of multiple sequence alignment. Comput Appl Biosci 11:13–18[Abstract]
  45. Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, Spellmeyer DC, Fox T, Caldwell JW, Kollman PA 1995 second generation force field for the simulation of proteins and nucleic acids. J Am Chem Soc 177:5179–5197[CrossRef]
  46. Sippl MJ 1993 Recognition of errors in three-dimensional structures of proteins. Proteins 17:355–362[Medline]
  47. Sanchez R, Sali A 1998 Large-scale protein structure modeling of the Saccharomyces cerevisiae genome. Proc Natl Acad Sci USA 95:13597–13602[Abstract/Free Full Text]
  48. Laskowski RA, MacArthur MW, Moss DS, Thornton JM 1993 PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26:283–291[CrossRef]
  49. Morris AL, MacArthur MW, Hutchinson EG, Thornton JM 1992 Stereochemical quality of protein structure coordinates. Proteins 12:345–364[Medline]
  50. Halgren TA 1996 Merck molecular force field. I. Basis, form, scope, parametrization, and performance of MMFF94. J Comput Chem 17:490–519[CrossRef]
  51. Halgren TA 1996 Merck molecular force field. II van der Waals and electrostatic parameters for intermolecular interactions. J Comput Chem 17:520–552[CrossRef]
  52. Wang R, Lai L, Wang S 2002 Further development and validation of empirical scoring functions for structure-based binding affinity prediction. J Comp Aided Mol Des 16:11–26[CrossRef]
  53. Ohno K, Fukushima T, Santa T, Waizumi N, Tokuyama H, Maeda M, Imai K 2002 Estrogen receptor binding assay method for endocrine disruptors using fluorescence polarization. Anal Chem 74:4391–4396[CrossRef][Medline]
  54. Westerfield M 2000 The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio), 4th Ed. Eugene: University of Oregon Press




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