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Applied and Environmental Microbiology, December 2005, p. 8995-8997, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8995-8997.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Rapid Detection of Subtype-Selective Nuclear Hormone Receptor Binding with Bacterial Genetic Selection

Georgios Skretas¹ and David W. Wood^1,2*

Department of Chemical Engineering,¹ Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544²

Received 12 July 2005/ Accepted 12 September 2005

Top Abstract Introduction References

 
Subtype-selective nuclear hormone receptor modulators could potentially allow the development of valuable tissue-specific therapeutics. A simple biosensor that allows subtype-specific nuclear hormone receptor binding to be reflected by the growth phenotype of Escherichia coli cells has been constructed. This system will potentially enable the facile detection or evolution of subtype-selective hormone analogues.

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The nuclear hormone receptors are the largest class of eukaryotic transcription factors and are present in all higher animal species (18). They are involved in cell development, differentiation, homeostasis, and metabolism (6). Failure to successfully execute their physiological tasks leads to a number of serious conditions, including carcinogenesis, infertility, obesity, inflammations, and osteoporosis; thus, they comprise one of the most important classes of protein pharmaceutical targets (2). Their activation is usually initiated by the binding of small-molecule hormones or hormone-mimicking compounds. A number of the members of this receptor superfamily exist in two or more subtypes (6). The estrogen receptor (ER), for example, exists in mammals in two different subtypes, termed ER and ERß. The ligand-binding domains of the two subtypes are evolutionarily very closely related, and they are activated similarly by natural estrogen (11). However, these domains have diverged sufficiently to allow certain naturally occurring or synthetic compounds to preferentially bind to and/or modulate only one particular subtype (11, 14, 16, 17). The observation that different receptor subtypes have variable tissue distributions in the human body (4, 5, 13) has attracted considerable attention to the discovery of subtype-selective hormone mimics. These compounds might potentially be used as tissue-specific therapeutics or as functional probes for the different receptor subtypes (1, 5, 7, 12). Simple, rapid, and economical systems with the ability to report subtype-specific behavior in a high-throughput format will become important tools for the discovery of such compounds.

In an earlier work, we constructed a simple sensor of estrogen binding by fusing the ER ligand-binding domain, in combination with a maltose-binding protein and an intein splicing domain, to a highly sensitive thymidylate synthase (TS) reporter enzyme (15). The activity of the TS reporter is increased upon ligand binding by the ER domain, thus producing readily detectable phenotype changes in TS-knockout Escherichia coli strains.

In this work, an ERß-based sensor was constructed using a strategy similar to that of the prototype estrogen-sensing vector pMIT::ER* (15) (hereafter referred to as pMIT::ER*) (Fig. 1A). Briefly, an artificial mini-intein was derived through the genetic deletion of the native endonuclease domain between residues 110 and 383 of the full-length Mycobacterium tuberculosis RecA intein, as described previously (3). The first amino acid of the intein also was mutated from cysteine to alanine in order to prevent splicing. The coding sequence corresponding to residues Arg254 to Lys504 of the human ERß was amplified from the plasmid pSG5-hERß (a gift from Cathleen Valentine, Department of Medicine, University of California, San Francisco, CA) and inserted into the unique BssHII site of the mini-intein to form a single open reading frame. This mini-intein/ERß fusion was then used to replace the mini-intein/ER fusion of the pMIT::ER* plasmid by utilizing the unique AgeI and XhoI restriction sites of the intein sequence. The resulting plasmid is referred to as pMIT::ERß* (Fig. 1A).

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FIG. 1. Estrogen-sensing protein designs and resulting growth phenotypes. (A) Domain fusions used to link estrogen binding via ER and ERß with thymidylate synthase activity. Ptac, artificial tac promoter containing a single-base-pair mutation required for hormone-dependent phenotypes (15); MBP, maltose-binding protein; N-Mtu, the first 96 amino acids of the M. tuberculosis RecA intein (Mtu RecA intein); C-Mtu, the last 41 amino acids of the Mtu RecA intein; N-Mtu', the first 110 amino acids of the Mtu RecA intein; C-Mtu', the last 58 amino acids of the Mtu RecA intein; ER, ligand-binding domain of human estrogen receptor ; ERß, ligand-binding domain of human estrogen receptor ß; TS, bacteriophage T4 thymidylate synthase enzyme. (B) Effect of the addition of 10 µM estrogen (E2) on the growth rates of cells carrying the pMIT::ERß* plasmid in liquid thymineless medium at 34°C. (C) Dose-response curve of cells transfected with pMIT::ERß* and grown in liquid thymineless medium at 34°C in the presence of E2 for approximately 15 h. Experiments were carried out in triplicate, and the error bars represent one standard deviation from the mean value. OD600, optical density at 600 nm.

As with the ER sensor (15), E. coli D1210thyA cells transfected with pMIT::ERß* are able to survive in liquid thymineless medium at 34°C only in the presence of 17-ß-estradiol (E₂; Sigma, St. Louis, MO) (Fig. 1B). By incubating cells in the presence of increasing concentrations of E₂, a dose-response curve was generated; it revealed a half-maximal effective concentration (EC₅₀) of approximately 100 nM and an apparent detection limit of 1 nM (Fig. 1C).

To assess the ability of this system to recognize subtype-selective ligand binding, E. coli D1210thyA cells were transfected with either pMIT::ER* or pMIT::ERß* and grown in liquid thymineless medium in the presence of a number of natural or synthetic estrogenic compounds. These compounds were chosen for their known preferential binding and/or receptor activation properties for one particular estrogen receptor subtype. We observed that the addition of the synthetic ER-selective compounds propylpyrazole triol (PPT) (16) and methyl piperidinopyrazole (MPP; Tocris Cookson, Ellisville, MO) (17) (Fig. 2A) enhanced cell growth only in cultures harboring the pMIT::ER* plasmid (Fig. 2B). On the other hand, the ERß-selective synthetic estrogen diarylpropionitrile (DPN; Tocris Cookson, Ellisville, MO) (14) and the ERß-selective phytoestrogens genistein and daidzein (Sigma, St. Louis, MO) (11) (Fig. 2A) produced much more pronounced growth enhancement in cells transfected with pMIT::ERß* (Fig. 2B). The presence of the nonselective natural estrogen E₂ led to a similar enhancement of growth for both sensor types.

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FIG. 2. Subtype-selective ligand binding and its effect on growth phenotypes. (A) Chemical structures of the estrogen analogues used in this study. (B) Cells transfected with either pMIT::ER* or pMIT::ERß* and grown in the presence of 10 µM natural or synthetic subtype-selective estrogenic compounds. The numbers above the growth bars are the ratios of the relative binding affinity of a particular compound for ER (RBA) to that for ERß (RBAß). RBA values are taken from works by Kim et al. (10) and Kuiper et al. (11). Cells were grown in liquid thymineless medium at 34°C for approximately 15 h. Experiments were carried out in triplicate, and the error bars represent one standard deviation from the mean value. OD600, optical density at 600 nm.

The efficiency of this bacterial biosensor to detect subtype-selective hormone binding was further demonstrated by exposing cells harboring either pMIT::ER* or pMIT::ERß* to increasing concentrations of subtype-specific estrogenic compounds (Fig. 3). The addition of PPT and MPP to cultures expressing the ER sensor resulted in TS activation and enhancement of cell growth at concentrations of around 1 µM, while no growth could be observed for cells expressing the ERß sensor, even at concentrations of as high as 10 µM. In contrast, DPN could activate the ERß sensor efficiently but was unable to enhance growth of cells carrying pMIT::ER* substantially. Weak DPN-induced TS activation via ER occurred at concentrations of around 10 µM, but full synthase activation could not be observed, as this hormone analogue became toxic for our bacterial sensor strains at higher concentrations. Similarly, the ERß-selective plant estrogen genistein exhibited a higher TS-activating and growth-enhancing potency for cells harboring pMIT::ERß*.

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FIG. 3. Cells harboring either pMIT::ER* (squares) or pMIT::ERß*(circles) and grown in the presence of increasing concentrations of the ER-selective compounds PPT and MPP and the ERß-selective estrogen analogues DPN and genistein. A four-parameter sigmoidal fit performed with KaleidaGraph software was used to generate the corresponding dose-response curves. Cells were grown in liquid thymineless medium at 34°C for approximately 15 h. Experiments were carried out in triplicate, and the error bars represent one standard deviation from the mean value. OD600, optical density at 600 nm.

These results are in many ways qualitatively consistent with those from ligand-binding assays based on estrogen receptor protein microarrays with or without coactivator recruitment (9, 10) as well as with some reporter gene transactivation assays of mammalian cells (10, 14, 16). For example, it has been shown that although PPT can bind to ERß (9), it is unable to activate this particular receptor subtype (10, 16). Furthermore, despite the higher binding specificity that genistein exhibits for ERß (approximately 500 times higher) than for the subtype specificity of DPN (approximately 100 times higher) (10), DPN has been found to be a much more subtype-selective estrogen receptor modulator (9, 10). The qualitative similarities of our constructed biosensor with more complex reporters of subtype-selective receptor binding are remarkable, considering the simplicity of this E. coli-based system. We have previously shown that our estrogen-sensing fusion protein can be trivially converted to a sensor of ligand binding for a different nuclear hormone receptor by simple binding domain swapping (15). It is therefore anticipated that the approach described here will be applicable to the search for subtype-specific hormone analogues for other members of the nuclear hormone receptor superfamily for which multiple subtypes exist. These include the thyroid hormone receptor, the retinoic acid receptor, and the peroxisome proliferator-activated receptor (PPAR) (6).

A number of systems with the ability to detect compounds with hormone-mimicking behavior have been developed (8, 19). However, most of them are not simple enough to enable rapid high-throughput screening of large numbers of products derived from combinatorial organic synthesis. The simplicity and speed of the described system promise to make it a valuable tool for the discovery of subtype-selective nuclear hormone receptor modulators with potential tissue-specific therapeutic properties. ERß-specific estrogen agonists or ER-selective antagonists, for example, could be used for the treatment of benign prostatic hyperplasia (7) without affecting male sexual behavior and fertility, while selective PPAR modulators could be used for the treatment of type 2 diabetes mellitus (1). Furthermore, because it utilizes bacterial genetic selection, the constructed biosensor constitutes a very facile technique for the in vivo evolution of small-molecule or peptide-based subtype-specific estrogenic compounds.

 
We thank Cathleen Valentine for supplying plasmids with the coding sequence for ERß.

This work was supported by National Science Foundation CAREER Award BES-0348220.

 
* Corresponding author. Mailing address: Department of Chemical Engineering, Princeton University, Engineering Quadrangle, Olden St., Princeton, NJ 08544. Phone: (609) 258-5721. Fax: (609) 258-0211. E-mail: dwood{at}princeton.edu.

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Applied and Environmental Microbiology, December 2005, p. 8995-8997, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8995-8997.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Skretas, G. Articles by Wood, D. W. Search for Related Content PubMed PubMed Citation Articles by Skretas, G. Articles by Wood, D. W. Agricola Articles by Skretas, G. Articles by Wood, D. W.