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High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays.

Fiebitz A, Nyarsik L, Haendler B, Hu YH, Wagner F, Thamm S, Lehrach H, Janitz M, Vanhecke D - BMC Genomics (2008)

Bottom Line: We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of DNA microarrays.Adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter.Moreover, different strategies in respect to bait-prey combinations are presented.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Planck Institute for Molecular Genetics, Department Vertebrate Genomics, Fabeckstr, 60-62, 14195 Berlin, Germany. fiebitz@molgen.mpg.de

ABSTRACT

Background: Most of the biological processes rely on the formation of protein complexes. Investigation of protein-protein interactions (PPI) is therefore essential for understanding of cellular functions. It is advantageous to perform mammalian PPI analysis in mammalian cells because the expressed proteins can then be subjected to essential post-translational modifications. Until now mammalian two-hybrid assays have been performed on individual gene scale. We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of DNA microarrays.

Results: In this cell array protein-protein interaction assay (CAPPIA), mixtures of bait and prey expression plasmids together with an auto-fluorescent reporter are immobilized on glass slides in defined array formats. Adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter. Using known interaction partners and by screening 160 different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. Moreover, different strategies in respect to bait-prey combinations are presented.

Conclusion: We demonstrate that the CAPPIA assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. The high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.

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Application of CAPPIA for the detection of hormone-dependent interactions. Triplicate slides each carrying 208 features, including all possible combinations of the 16 prey and 10 bait proteins listed in Additional file 1 and sets of positive (p53+SV40T) and negative (p53+TRAF, single baits and preys) controls were reverse transfected with Hek293T cells in the presence of 10 nM R1881 for 3 days. Each prey-bait combination was printed together with the autofluorescent reporter as single spots per slide with a spot to spot distance of 1.5 mm. Following transfection the fluorescence signals of all 624 features were collected and processed as described in methods. The normalised fluorescence signals obtained from the 480 different bait-prey features (10 baits × 16 preys × 3 replicate slides) are shown separately for the different slides. Sample numbers correspond to the combination of bait and prey as summarized in Additional file 1. The cutoff value (indicated as red line) resembles the level of reporter expression obtained with the non-interacting control proteins p53+TRAF. This cross-screening using CAPPIA immediately identifies so-called bait-or prey-unspecific false positives as is exemplified by bait B504 (marked with a star). A specific interaction was identified between B487 and P506 corresponding to the AR-LBD and AR-NTD (sample number 23, marked with an arrow).
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Figure 2: Application of CAPPIA for the detection of hormone-dependent interactions. Triplicate slides each carrying 208 features, including all possible combinations of the 16 prey and 10 bait proteins listed in Additional file 1 and sets of positive (p53+SV40T) and negative (p53+TRAF, single baits and preys) controls were reverse transfected with Hek293T cells in the presence of 10 nM R1881 for 3 days. Each prey-bait combination was printed together with the autofluorescent reporter as single spots per slide with a spot to spot distance of 1.5 mm. Following transfection the fluorescence signals of all 624 features were collected and processed as described in methods. The normalised fluorescence signals obtained from the 480 different bait-prey features (10 baits × 16 preys × 3 replicate slides) are shown separately for the different slides. Sample numbers correspond to the combination of bait and prey as summarized in Additional file 1. The cutoff value (indicated as red line) resembles the level of reporter expression obtained with the non-interacting control proteins p53+TRAF. This cross-screening using CAPPIA immediately identifies so-called bait-or prey-unspecific false positives as is exemplified by bait B504 (marked with a star). A specific interaction was identified between B487 and P506 corresponding to the AR-LBD and AR-NTD (sample number 23, marked with an arrow).

Mentions: Having demonstrated the specificity and efficiency of CAPPIA, we next explored whether this method allows the large scale screening for hormone-dependent interactions. Steroid hormone receptors such as the androgen receptor (AR) are ligand-dependent transcription factors that control a variety of essential physiologic and developmental processes in humans [14-17]. Here, in the presence of 10 nM of the synthetic androgenic ligand R1881, we analysed the interaction of 10 individual baits with 16 different preys coding for proteins or protein domains potentially associated with nuclear receptor function (Additional file 1). Each slide contained all 160 different prey-bait combinations as single features in addition to a series of positive and negative controls to localise the array and monitor transfection efficiency as well as background fluorescence. In total each slide contained 208 spots, and three identical slides were transfected to obtain independent replicate data points for each prey-bait combination. Fluorescence signals of all 624 features were collected and processed as described in methods. The 480 data points representing reporter expression induced by the different prey-bait combinations are summarized in Figure 2. Bait B504 can immediately be recognized as being a so-called false positive bait since it activates reporter expression with multiple unrelated preys. Such false positives induce reporter expression either by unspecific binding or, as is the case for the fusion protein (AR-NTD) of bait B504, by conferring trans-activating activity to the GAL4 DNA binding domain of the bait [18]. More importantly, a specific and androgen-dependent interaction was identified between bait B487, the ligand binding domain of the AR (AR-LBD), and prey P506, the N-terminal domain of the AR (AR-NTD). This interaction was confirmed in a second CAPPIA experiment (Additional file 2).


High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays.

Fiebitz A, Nyarsik L, Haendler B, Hu YH, Wagner F, Thamm S, Lehrach H, Janitz M, Vanhecke D - BMC Genomics (2008)

Application of CAPPIA for the detection of hormone-dependent interactions. Triplicate slides each carrying 208 features, including all possible combinations of the 16 prey and 10 bait proteins listed in Additional file 1 and sets of positive (p53+SV40T) and negative (p53+TRAF, single baits and preys) controls were reverse transfected with Hek293T cells in the presence of 10 nM R1881 for 3 days. Each prey-bait combination was printed together with the autofluorescent reporter as single spots per slide with a spot to spot distance of 1.5 mm. Following transfection the fluorescence signals of all 624 features were collected and processed as described in methods. The normalised fluorescence signals obtained from the 480 different bait-prey features (10 baits × 16 preys × 3 replicate slides) are shown separately for the different slides. Sample numbers correspond to the combination of bait and prey as summarized in Additional file 1. The cutoff value (indicated as red line) resembles the level of reporter expression obtained with the non-interacting control proteins p53+TRAF. This cross-screening using CAPPIA immediately identifies so-called bait-or prey-unspecific false positives as is exemplified by bait B504 (marked with a star). A specific interaction was identified between B487 and P506 corresponding to the AR-LBD and AR-NTD (sample number 23, marked with an arrow).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254387&req=5

Figure 2: Application of CAPPIA for the detection of hormone-dependent interactions. Triplicate slides each carrying 208 features, including all possible combinations of the 16 prey and 10 bait proteins listed in Additional file 1 and sets of positive (p53+SV40T) and negative (p53+TRAF, single baits and preys) controls were reverse transfected with Hek293T cells in the presence of 10 nM R1881 for 3 days. Each prey-bait combination was printed together with the autofluorescent reporter as single spots per slide with a spot to spot distance of 1.5 mm. Following transfection the fluorescence signals of all 624 features were collected and processed as described in methods. The normalised fluorescence signals obtained from the 480 different bait-prey features (10 baits × 16 preys × 3 replicate slides) are shown separately for the different slides. Sample numbers correspond to the combination of bait and prey as summarized in Additional file 1. The cutoff value (indicated as red line) resembles the level of reporter expression obtained with the non-interacting control proteins p53+TRAF. This cross-screening using CAPPIA immediately identifies so-called bait-or prey-unspecific false positives as is exemplified by bait B504 (marked with a star). A specific interaction was identified between B487 and P506 corresponding to the AR-LBD and AR-NTD (sample number 23, marked with an arrow).
Mentions: Having demonstrated the specificity and efficiency of CAPPIA, we next explored whether this method allows the large scale screening for hormone-dependent interactions. Steroid hormone receptors such as the androgen receptor (AR) are ligand-dependent transcription factors that control a variety of essential physiologic and developmental processes in humans [14-17]. Here, in the presence of 10 nM of the synthetic androgenic ligand R1881, we analysed the interaction of 10 individual baits with 16 different preys coding for proteins or protein domains potentially associated with nuclear receptor function (Additional file 1). Each slide contained all 160 different prey-bait combinations as single features in addition to a series of positive and negative controls to localise the array and monitor transfection efficiency as well as background fluorescence. In total each slide contained 208 spots, and three identical slides were transfected to obtain independent replicate data points for each prey-bait combination. Fluorescence signals of all 624 features were collected and processed as described in methods. The 480 data points representing reporter expression induced by the different prey-bait combinations are summarized in Figure 2. Bait B504 can immediately be recognized as being a so-called false positive bait since it activates reporter expression with multiple unrelated preys. Such false positives induce reporter expression either by unspecific binding or, as is the case for the fusion protein (AR-NTD) of bait B504, by conferring trans-activating activity to the GAL4 DNA binding domain of the bait [18]. More importantly, a specific and androgen-dependent interaction was identified between bait B487, the ligand binding domain of the AR (AR-LBD), and prey P506, the N-terminal domain of the AR (AR-NTD). This interaction was confirmed in a second CAPPIA experiment (Additional file 2).

Bottom Line: We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of DNA microarrays.Adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter.Moreover, different strategies in respect to bait-prey combinations are presented.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Planck Institute for Molecular Genetics, Department Vertebrate Genomics, Fabeckstr, 60-62, 14195 Berlin, Germany. fiebitz@molgen.mpg.de

ABSTRACT

Background: Most of the biological processes rely on the formation of protein complexes. Investigation of protein-protein interactions (PPI) is therefore essential for understanding of cellular functions. It is advantageous to perform mammalian PPI analysis in mammalian cells because the expressed proteins can then be subjected to essential post-translational modifications. Until now mammalian two-hybrid assays have been performed on individual gene scale. We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of DNA microarrays.

Results: In this cell array protein-protein interaction assay (CAPPIA), mixtures of bait and prey expression plasmids together with an auto-fluorescent reporter are immobilized on glass slides in defined array formats. Adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter. Using known interaction partners and by screening 160 different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. Moreover, different strategies in respect to bait-prey combinations are presented.

Conclusion: We demonstrate that the CAPPIA assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. The high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.

Show MeSH
Related in: MedlinePlus