A GAL4-based yeast three-hybrid system for the identification of small molecule–target protein interactions

Henthorn, Debbie C.; Jaxa‐Chamiec, Albert; Meldrum, Eric

doi:10.1016/s0006-2952(02)00884-5

Cited by 53 publications

(45 citation statements)

References 17 publications

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“…Because it relies on the internal coexpression of "bait" and "prey" fusion proteins, the twohybrid system cannot be used to identify proteins that bind to externally synthesized or modified proteins or compounds. More recently, variations of the two-hybrid system have been developed for the analysis of post-translational modificationdependent protein-protein interactions and small moleculeprotein interactions (16,17). However, these methods require the design and construction of assay-specific components and face uncertainties in efficiency and specificity of modifications that take place inside the yeast cell (16).…”

mentioning

confidence: 99%

Construction and Application of a Yeast Surface-displayed Human cDNA Library to Identify Post-translational Modification-dependent Protein-Protein Interactions

Bidlingmaier

Liu

2006

Molecular & Cellular Proteomics

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Although post-translational modifications such as phosphorylation mediate fundamental biological processes within the cell, relatively few methods exist that allow proteome-wide identification of proteins that interact with these modifications. We constructed a yeast surface-displayed human cDNA library and utilized it to identify protein fragments with affinity for phosphorylated peptides derived from the major tyrosine autophosphorylation sites of the epidermal growth factor receptor or focal adhesion kinase. We identified cDNAs encoding the Src homology 2 domains from adapter protein APS, phosphoinositide 3-kinase regulatory subunit 3, SH2B, and tensin, demonstrating the effectiveness of this approach.

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mentioning

confidence: 99%

Construction and Application of a Yeast Surface-displayed Human cDNA Library to Identify Post-translational Modification-dependent Protein-Protein Interactions

Bidlingmaier

Liu

2006

Molecular & Cellular Proteomics

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“…Cells with the FK506-dexamethasome heterodimer showed reporter activity, while in cells treated with the heterodimer and free FK506, reporter activity was abrogated, demonstrating the utility of the screen for finding SM that can compete with the native receptor ligand. In another more recent study by Henthorn and co-workers, a similar setup was employed to screen a mouse cDNA library to identify targets of the SM methotrexate, which was covalently linked to dexamethasone [16]. As many as 56 protein targets were identified.…”

Section: Yeast Two-hybrid Systemmentioning

confidence: 99%

Target Profiling of Small Molecules

Chepelev

Dumontier

2009

Protein Targeting With Small Molecules

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“…Such systems have been adapted from the transcription factor-based yeast two-hybrid system. [2][3][4][5] A synthetic hybrid ligand serves as a chemical inducer of dimerization (CID) for the respective protein moieties fused to the DNA-binding or transactivation domain of a transcription factor. The successful three-hybrid formation is then monitored by reporter gene activation upon reconstitution of the transcription factor.…”

Section: Introductionmentioning

confidence: 99%

A Small‐Molecule–Protein Interaction System with Split‐Ubiquitin as Sensor

Dirnberger

Unsin²,

Schlenker³

et al. 2006

ChemBioChem

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The identification of receptors for small molecules is of great pharmaceutical importance for drug-discovery research. Several systems for the identification of protein-small-molecule interactions have been developed in the past. These were modifications of the classical yeast two-hybrid system, relying on a transcriptional read-out following nuclear translocation of the complex. Here we present a novel three-hybrid technology based on the split-ubiquitin system for the analysis of protein-small-molecule interactions independently of a nuclear translocation of the complex. The performance of the system is compared to a method based on the classical yeast two-hybrid system by using a chemical inducer of dimerization (CID) comprised of methotrexate linked to dexamethasone. Steric issues are addressed by varying the linker length of the compounds, as well as by comparing the orientation of fusion proteins. The system is further extended to the analysis of a small-molecule inhibitor of human PCTAIRE protein kinase 3, which is related to cyclin-dependent kinases (CDKs), an important class of pharmaceutical targets.

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A GAL4-based yeast three-hybrid system for the identification of small molecule–target protein interactions

Cited by 53 publications

References 17 publications

Construction and Application of a Yeast Surface-displayed Human cDNA Library to Identify Post-translational Modification-dependent Protein-Protein Interactions

Construction and Application of a Yeast Surface-displayed Human cDNA Library to Identify Post-translational Modification-dependent Protein-Protein Interactions

Target Profiling of Small Molecules

A Small‐Molecule–Protein Interaction System with Split‐Ubiquitin as Sensor

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