Detection of Peptides, Proteins, and Drugs That Selectively Interact With Protein Targets

I. G. Serebriiskii
2002 Genome Research  
Genome sequencing has been completed for multiple organisms, and pilot proteomic analyses reported for yeast and higher eukaryotes. This work has emphasized the facts that proteins are frequently engaged in multiple interactions, and that governance of protein interaction specificity is a primary means of regulating biological systems. In particular, the ability to deconvolute complex protein interaction networks to identify which interactions govern specific signaling pathways requires the
more » ... ration of biological tools that allow the distinction of critical from noncritical interactions. We report the application of an enhanced Dual Bait two-hybrid system to allow detection and manipulation of highly specific protein-protein interactions. We summarize the use of this system to detect proteins and peptides that target well-defined specific motifs in larger protein structures, to facilitate rapid identification of specific interactors from a pool of putative interacting proteins obtained in a library screen, and to score specific drug-mediated disruption of protein-protein interaction. [Supplemental material is available online at http://www.genome.org. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: A. Taliana, M. Russell, M. Berman, and R. Finley.] Since its inception (Fields and Song 1989), the two-hybrid system has been utilized in increasingly complex strategies to analyze interactions between proteins of biological interest and known or novel cognate partners including other proteins, RNA sequences, pharmacological agents, and peptides (for review, see . More recently, a number of groups have exploited the potential of two-hybrid systems as a tool for understanding protein interactions on a genome-level scale, with pilot studies involving elucidation of large sets of protein interactions developed in Saccharomyces cerevisiae (Schwikowski et al. 2000; Ito et al. 2001 ) providing a model for ongoing work in higher eukaryotes. Given the increasing realization that protein interaction networks involve the interaction of discrete signaling molecules with multiple partner proteins in different biological circumstances, accurate description of the function of a given protein now implicitly involves dissection of its interaction domains, ranking of its interaction affinity with each of its partners, and determination of physiological conditions under which each pair of proteins preferentially interacts. These determinations pose significant technological hurdles in high-throughput efforts. We have described previously a proof-of-concept experiment for a two-hybrid Dual Bait system that provides internal controls for interaction specificity, and could theoretically be used to selectively compare the interaction of a protein with more than one partner molecule (Serebriiskii et al. 1999) . Building from this preliminary study, we have now developed a complete system of reagents that can be used to score interaction of one transcriptional activation domain (AD)-fused prey protein with either of two DNA-binding domain (DBD)fused bait proteins over a range of different interaction affinities. In three different library screening applications, we demonstrate that these reagents can be used to identify proteins or peptides that target short sequence elements of biological importance within a larger protein structure. We further demonstrate that the system can be used in a bait swap application for rapid secondary screening to sort multiple library hits into subgroups most likely to be reproducible and physiologically relevant. Finally, we describe the use of the reagents in a subtractive two-color visualization procedure that can discriminate specific from nonspecific drug-induced inhibition of protein interactions. These studies, together with our other work involving use of the system to build enhanced specificity derivatives of signaling proteins engaged in complex interactions, indicate the Dual Bait is a useful tool in dissection of complex cellular regulatory machinery. RESULTS AND DISCUSSION In the Dual Bait two-hybrid system (Fig. 1) , the use of two parallel bait-reporter systems allows simultaneous and com-6 Corresponding author. E-MAIL EA_Golemis@fccc.edu; FAX (215) 728-3616. Article and publication are at
doi:10.1101/gr.450702 pmid:12421766 pmcid:PMC187545 fatcat:ulqmbgf3ijfdhobv4o5tapojvu