This system has been successfully applied to identify a novel glycosynthase (51)

This system has been successfully applied to identify a novel glycosynthase (51). MTX forms one half of yet another dimerizer system, this one developed by the Johnsson lab (52), which has linked it to anO6-benzylguanine (BG) derivative. efforts to bring proteins together to induce a specific biological effect. A bifunctional small molecule can be as simple as a natural product that interacts with two protein molecules simultaneously, such as the antibiotic coumermycin (observe below) or the fungal toxin brefeldin A, which inhibits the small G-protein Arf by stabilizing an inactive complex with GDP-Arf and a guanine nucleotide exchange factor (5). However, the majority of successful, synthetic, bifunctional molecules consist of two protein-binding moieties joinedviaa linker of appropriate length (Physique 1). If these moieties are identical (homobifunctional), the molecule will dimerize two identical protein monomers, but if the moieties differ (heterobifunctional), two different proteins can be brought together. The applications of these molecules are as varied as their design. For example, the juxtaposition of two or more proteins can cause ligand-independent activation of receptors or bipartite transcription factors. It can sequester one protein in the subcellular location of the other, target a protein for destruction, or produce a binding surface recognized by other proteins, leading to positive or unfavorable regulation of signaling. Bifunctional molecules can specifically localize toxins, and drug hybrids (observe below) can influence the activity of two proteins without forming dimers. Notably, in most cases these effects are tunable (by varying small molecule concentration), and reversible (on removal of drug). == Physique 1. == A generic bifunctional molecule brings two protein molecules together to induce a biological effect. The concept of chemical inducers of dimerization (CID) (6) dates to early work by the Schreiber and Crabtree groups (7). These labs required advantage of the specific binding of the natural product FK506 to the PRIMA-1 protein FKBP12 (FK506 binding protein 12). Normally, this drug acts as a natural heterodimerizer, causing the recruitment of the protein phosphatase calcineurin to the FK506-FKBP12 complex. However, an artificial dimer of FK506 named FK1012 instead promotes FKBP12 homodimerization, allowing small-molecule controlled dimerization of signaling proteins (in the beginning, the cytoplasmic domains of the T-cell receptor subunit) fused to FKBP12 monomers (7). Similarly, PRIMA-1 the natural product heterodimerizer rapamycin promotes conversation between FKBP12 and the FKBP-rapamycin-binding (FRB) domain name of mTor. PRIMA-1 Thus, rapamycin can be used to heterodimerize protein fusions made up of FKBP and FRB domains (8). Alternatively, heterodimerization can be induced with an FK506-cyclosporin fusion, which brings together FKBP12 and cyclophilin proteins (9). Subsequently, variants of these molecules were designed to bind mutant versions of their target proteins, creating orthogonal dimerizer systems that have been used in transgenic mice (10,11), although recent evidence suggests that both mutant and wild-type FRB-protein fusions are destabilized (12). Dimerization packages based on these systems Rabbit Polyclonal to JAK2 (phospho-Tyr570) have been developed by ARIAD Pharmaceuticals, Inc (http://www.ariad.com/wt/page/regulation_kits). The diversity of applications of FKBP-based CID has PRIMA-1 been comprehensively reviewed recently (13). One noteworthy variant is the yeast three-hybrid system (Physique 2) for identifying protein targets of a small molecule (examined in (14)). Similar to the yeast two-hybrid method for detecting protein-protein interactions, this approach entails bringing a transcriptional activation domain name fusion into contact with a DNA-binding domain name fusion, leading to transcription of a reporter gene. Rather than relying on protein-protein interactions of the two fused proteins to activate transcription, the three-hybrid approach uses a CID, with a moiety that binds to the DNA-binding domain name fusion, and a moiety that binds to the activation domain name fusion. If the latter moiety is a small molecule of unknown target, a library of activation domain-cDNA fusions can be used to identify possible targets. The first proof-of-principle of this system used the synthetic glucocorticoid dexamethasone fused to FK506 to bring FKBP12 and glucocorticoid receptor fusions.