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

Abstract

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.

Signaling of Human Frizzled Receptors to the Mating Pathway in Yeast

“…pP CUP1 -Fz1-CRU: This construct is based on the previously described vector pP CUP1 -ubc9-CRU [45], which is a low copy yeast - E. coli shuttle vector that carries a C ub -R-URA3 cassette (CRU), a copper-dependent promoter (P CUP1 ) for bait expression, and a HIS3 marker. The human Frizzled 1 coding sequence was amplified by PCR from a human whole brain cDNA preparation (Clontech) using primers Fz1-FW (5′-AATTGTCGACATGGCTGAGGAGGAGGCGCCTAAG-3′) and Fz1-RV (5′-AGCGGCCGCGACTGTAGTCTCCCCTTGTTTGC-3′), introducing Sal I and Not I restriction sites, respectively.…”

Techniques for the analysis of protein-protein interactions in vivo

“…Hence, the URA3-based splitubiquitin system can be used for either the detection of binary PPIs (when counterselection with 5-fluoroorotic acid is used) or the screening of inhibitors or point mutations that abolish an interaction (when using uracildepleted medium for selection). While the cost for the chemical compound 5-fluoroorotic acid and the necessary replica plating represent downsides, this reporter system facilitates screening for inhibitors of an interaction at the membrane or in the cytosol or nucleus (Dirnberger et al, 2006).…”

Split‐Ubiquitin and the Split‐Protein Sensors: Chessman for the Endgame

“…Supplying the growth media with this CID allowed the yeast cells co-expressing the corresponding split-Ub fusion proteins to grow on media containing 5-FOA as a positive indicator of the binding of dexamethasone to GR. [55] The advantage of the split-Ub system over the twohybrid approach is its flexibility with regard to the type of proteins that can be tested against small-molecule interactions A C H T U N G T R E N N U N G including membrane-associated receptors or channels. This expanded applicability is of considerable interest as many of the potential targets for synthetic compound screens are residents of this compartment.…”

“…[ 55] In a proof of principle study, N ub was fused to dihydrofolate reductase (DHFR) and the C ub -R-Ura 3 p module was coupled to the rat gluticocorticoid receptor (GR). Accordingly, the hybrid CIP consisted of methotrexate as the ligand for DHFR coupled to dexametasone as the ligand for GR.…”