# Strategies for Post-Translational Control of Protein Expression and Their Applications
## 4 Conditional Control of Protein Stability Using Genetic Manipulation
### Small-Molecule-Switchable Degrons  
The representative work of small-molecule-based degron technology is the destabilizing domain (DD) developed by the [PERSON] group. An engineered variant of the human FK506- and rapamycin-binding protein (FKBP12), FKBP12 F36V, possesses a \"hole\" that allows a \"bumped\" ligand, Shield-1, to bind more tightly to the F36V mutant compared with the wild-type protein, by almost three orders of magnitude. Using random mutagenesis, they identified mutants of FKBP12 F36V that are unfolded, ubiquitinated, and rapidly and constitutively degraded when expressed in mouse and human-derived cells [38]. These domains are called destabilizing domains (DDs), and this instability is conferred to other proteins fused to these DDs. The addition of Shield-1, which binds to the FKBP12 DD and stabilizes the DD protein fold, rescues them from degradation, allowing fusion proteins to perform their functions (Figure 3a). This \"drug-on\" system is reversible and tunable, as well as useful for the study of constitutively active enzymes. Those authors further created orthogonal DDs using a combination of _Escherichia coli_ dihydrofolate reductase [39], the human estrogen receptor ligand-binding domain [40], or the bilirubin-binding protein (UnaG) [41], and their ligands. In addition to reversibility and tunability, the drug-dependent stabilization of the target protein is an important feature of the DD technology. It has been shown that the DD approach works in several organisms [42; 43; 44; 45; 46; 47]. Although this is useful for the study of constitutively active enzymes, etc., it requires the continuous exposure of the stabilizing ligand to the cells, for protein expression. The authors established a complementary technique with a ligand-induced degradation (LID) domain in which a 19-amino-acid degron is fused to the C terminus of FKBP12 F36V (Figure 3b) [48]. In the absence of Shield-1, the degron is bound to FKBP12 F36V and the fusion protein is stable. In turn, the addition of Shield-1, which binds to FKBP12 F36V and displaces the degron, induces the degradation of the fusion protein. Fusion proteins with tags can be targeted for degradation via recruitment to specific E3 Ub ligases using chimeric molecules such as PROTAC and SNIPER. These techniques are based on the inducible proximity between the POI and the E3 ligase using a Halo tag, which is a modified haloalkene dehalogenase designed to covalently bind to the synthetic ligand (Halo tag ligand). The [PERSON] group developed HaloPROTACs that contain a ligand for HaloTag conjugated to POIs and a ligand for the VHL E3 Ub ligase [49]. HaloPROTACs bind to both the HaloTag conjugated to POIs and the VHL E3 Ub ligase, leading to the ubiquitination and degradation of the fusion protein with POIs (Figure 3d). This group also developed a hydrophobic tagging (Hyt) strategy using the HaloTag fusion protein system [50]. A hydrophobic group of the misfolded protein is exposed on its surface, to be recognized by the 70-kDa heat-shock protein (HSP70), which specifically binds hydrophobic amino acids and helps the folding of slow-folding proteins (Figure 3c) [51]. When a misfolded protein is not properly folded into its native structure by HSP70, the complex of HSP70 and substrate is recognized by CHIP, which is a chaperone-dependent E3 Ub ligase, resulting in degradation. Based on the mechanism in which a protein carrying a hydrophobic tag can mimic the misfolded protein, they designed and synthesized a small-molecule with an adamantyl moiety to bind a HaloTag protein. A HaloTag fusion protein is degraded by addition of the hydrophobic-tagged molecule with adamantly moiety. In addition, [PERSON] et al. used the HyT method to reduce the level of the microtubule-associated protein Tau, which is related to [PERSON]'s disease [52]. They designed and synthesized a chimeric molecule that consists of an adamantyl moiety, a Tau-recognition motif, and a cell-penetrating peptide motif. Analogously, [PERSON] et al. developed a degradation tag (dTAG) system [53].