cDNA phage display as a novel tool to screen for cellular targets of chemical compounds
Introduction
In toxicology, a lot of research effort is devoted to the development of new scientific tools to provide a better insight in the mechanism of action of chemical compounds. To unravel the mechanism of action of chemical compounds, it is crucial to determine the cellular targets of these chemical compounds. So far cellular targets are often determined with proteomics tools, like affinity chromatography combined with mass spectrometry (Cekaite et al., 2007, Rix and Superti-Furga, 2009), cell screening (Bradner et al., 2006) and protein microarrays (MacBeath and Schreiber, 2000). Unfortunately, such identification of cellular targets is an expensive, slow and laborious process, especially if the cellular targets are expressed at low levels in their respective tissue. To circumvent the need of these expensive methods, many gene-linked methods have been developed, where there is a physical linkage between the phenotype and the genotype, including yeast and mammalian three-hybrid assays (Baker et al., 2003, Caligiuri et al., 2006, Kley, 2004) and bacterial and yeast surface display (Bidlingmaier and Liu, 2007, Zhang et al., 2008). Although all of these assays have been used successfully, the simplest and most widespread cost-effective system for executing this linkage is phage display technology (Scott and Barbas, 2001). With phage display it is possible to screen for proteins or peptides with affinity for chemical compounds. This can be performed by using phage libraries, which consist of a high number of different phages (108–1010) that are all displaying a different peptide or protein on their surface. Out of these phage libraries, the phages with affinity for a chemical compound can be selected with an affinity selection procedure. Moreover, the peptide or protein displayed on these selected phages can easily be identified by sequencing the gene that codes for the displayed protein. This coding gene is present in the phagemid DNA inside the selected phage. In drug development research, phage display is frequently used to screen for cellular targets of drugs (Jin et al., 2002, Makowski and Rodi, 2003, Mandava et al., 2004, Rodi et al., 1999, Rodi et al., 2001, Sche et al., 1999, Yu et al., 2007). However, in toxicology, phage display remains unexplored, in spite of its large potential in this field. In our previous paper, the potential of peptide phage display as a screening tool for cellular targets of heavy metals was demonstrated (Van Dorst et al., 2010). An important limitation of the described peptide phage display is that the binding properties of the peptides displayed on the phage surface must mimic the binding properties of similar sequences in cellular proteins. This is not always the case, since the three-dimensional configuration of a cellular protein is also important during binding of chemical compounds. However, with the cDNA phage display technique used in the current paper, it is possible to display cellular proteins in their proper three-dimensional configurations on the surface of the phage. Phages selected with cDNA phage display are also more relevant than those selected with peptide phage display. Moreover, the sequences of the displayed proteins of the selected cDNA phages are 100% identical to cellular proteins, whereas with peptide phage display, there has to be an overlap of the selected peptide with a cellular protein.
In the present study, the potential of cDNA phage display in toxicology as a screening tool for interactions between chemical compounds and cellular targets was demonstrated with bisphenol A (BPA) as a model compound. Cellular proteins with affinity for BPA were selected from a cDNA phage library of the colorectal cancer cell line HT-29. Besides the selection of a known cellular target of BPA, also a possible novel cellular target of BPA was selected. This demonstrates the potential of cDNA phage display as a novel tool to screen for interactions between chemical compounds and cellular targets.
Section snippets
Experimental section
Biotinylated BPA, that was used as model compound was kindly provided by Prof. Arterburn (New Mexico State University, USA). Fig. 1 shows the structure of this biotinylated BPA. The synthesis of this biotinylated BPA is described in George et al. (2008) (BPA-biotin 2 in the paper of George et al. (2008)). To immobilize the biotinylated BPA, streptavidin coated magnetic beads (dynabeads M280 streptavidin, Invitrogen) were used. The cDNA phage library was prepared by cloning a cDNA library of the
Results
To demonstrate the potential of cDNA phage display in toxicology as a screening tool for cellular targets of chemical compounds, cellular proteins with affinity for BPA were selected from a cDNA phage library of the colorectal cancer cell line HT-29. Therefore, a phage affinity selection procedure as illustrated in Fig. 2 was performed. For this affinity selection, biotinylated BPA was used as a target. The biotinylation of the BPA allows binding to streptavidin coated beads, essential to
Discussion
In the present study, the potential of cDNA phage display as a screening tool for interactions between chemical compounds and cellular targets was demonstrated, with BPA as a model compound. The selection of BPA as a model compound in this study was based on the extensive mechanistic information available in literature. This information is important as a reference for the evaluation of the identified cellular targets and subsequent evaluation of the cDNA phage display approach. For the
Acknowledgments
The present study was financially supported by the ‘Institute for the promotion of innovation by science and technology (IWT)’ in Flanders (Belgium) and by the Federal Public Service of Health, Food Chain Safety and Environment (contract RT 07/11 INVITRAB and RF6204 ERGOT). The authors like to thank Prof. Jeffrey B. Arterburn (New Mexico State University) for providing the biotinylated BPA.
References (38)
- et al.
The estradiol pharmacophore: ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site
Steroids
(1997) - et al.
An optimized dexamethasone-methotrexate yeast 3-hybrid system for high-throughput screening of small molecule–protein interactions
Anal. Biochem.
(2003) - et al.
Interrogating yeast surface-displayed human proteome to identify small molecule-binding proteins
Mol. Cell Proteomics
(2007) - et al.
A robust small-molecule microarray platform for screening cell lysates
Chem. Biol.
(2006) - et al.
MASPIT: three-hybrid trap for quantitative proteome fingerprinting of small molecule–protein interactions in mammalian cells
Chem. Biol.
(2006) - et al.
Phage display of cDNA repertoires: the pVI display system and its application for the selection of immunogenetic ligands
J. Immunol. Methods
(1999) - et al.
Bisphenol a exposure causes meiotic aneuploidy in the female mouse
Curr. Biol.
(2003) - et al.
Identification of hNopp140 as a binding partner for doxorubicin with a phage display cloning method
Chem. Biol.
(2002) - et al.
Mechanistic investigations of low dose exposures to the genotoxic compounds bisphenol-A and rotenone
Mutat. Res.
(2008) Chemical dimerizers and three-hybrid systems: scanning the proteome for targets of organic small molecules
Chem. Biol.
(2004)
Bisphenol A and its methylated congeners inhibit growth and interfere with microtubules in human fibroblasts in vitro
Chem. Biol. Interact.
Induction of multiple microtubule-organizing centers, multipolar spindles and multipolar division in cultured V79 cells exposed to diethylstilbestrol, estradiol-17beta and bisphenol A
Mutat. Res.
Interference with microtubules and induction of micronuclei in vitro by various bisphenols
Mutat. Res.
Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol-binding protein
J. Mol. Biol.
Display cloning: functional identification of natural product receptors using cDNA-phage display
Chem. Biol.
Flow cytrometic cell cycle analysis allows for rapid screening of estrogenicity in MCF 7 breast cancer cells
Toxicol. In Vitro
Screening of phage displayed human liver cDNA library against dexamethasone
J. Pharm. Biomed. Anal.
Convergent transcriptional profiles induced by endogenous estrogen and distinct xenoestrogens in breast cancer cells
Carcinogenesis
Protein arrays: a versatile toolbox for target identification and monitoring of patient immune responses
Methods Mol. Biol.
Cited by (22)
Identification of novel paraben-binding peptides using phage display
2020, Environmental PollutionFoliar graphene oxide treatment increases photosynthetic capacity and reduces oxidative stress in cadmium-stressed lettuce
2020, Plant Physiology and BiochemistryCitation Excerpt :Chlorophyll fluorescence parameters are closely related to the photosynthetic processes of plant mesophyll cells (Hussain and Reigosa, 2011). Several parameters—including Fv/Fm, ϕPSII, and ETR— are widely used to study the effects of environmental stress on plants (Van Dorst et al., 2010). In our study, lettuce plants grown with Cd2+ alone had lower Fv/Fm, ϕPSII, and ETR values than control plants, indicating that photosynthesis was inhibited.
Identification of UQCRB as an oxymatrine recognizing protein using a T7 phage display screen
2016, Journal of EthnopharmacologyTarget deconvolution techniques in modern phenotypic profiling
2013, Current Opinion in Chemical BiologyEffects of bisphenol A on growth, photosynthesis and chlorophyll fluorescence in above-ground organs of soybean seedlings
2013, ChemosphereCitation Excerpt :Chlorophyll fluorescence emitted by higher plants can reflect photosynthetic activities in a complex manner (Hussain and Reigosa, 2011). The F0, Fv/Fm, ΦPSII and ETR are the more representative chlorophyll fluorescence parameters, and thus they are widely used in the studies on the effects of environmental stress on plants (El-Khatib et al., 2004; Haldimann and Feller, 2004; Hajek and Skopal, 2009; Abdeshahian et al., 2010; Van Dorst et al., 2010). The F0 is the fluorescent when the reaction center of photosystem II (PSII) are all open, and the increase in F0 indicates the injury of PSII (Kitajima and Butler, 1975).