Research summary

Technology to conditionally regulate protein stability without interfering the function is a powerful tool for biomedical research. Recently, several research groups have developed a variety of methodologies for control of expression level of the target protein using a degron peptide, which confers instability to tagged protein and induce the proteasomal degradation in response to chemical treatment or photo irradiation. Unlike RNAi and Cre/loxP systems, these methods regulate the protein stability in post-translational level and enable to control the protein expression within 30-60 min after the stimulation. However, the most of degron systems require permanent fusion of targeted protein with a degron tag, which may disrupt the molecular function of the target in some cases. Previously we have developed a complementary method to conditionally control cellular protein stability by introducing a ubiquitin variant between destabilizing domain (DD) and targeted protein. In the absence of the stabilizing ligand, the DD dominates and the entire polypeptide is rapidly degraded by the ubiquitin-proteasome system. In the presence of stabilizing ligand, the fusion protein is stabilized and becomes a substrate for abundant ubiquitin-specific proteases (informally called deubiquitinating enzyme or DUB), liberating a native or a near-native protein of interest (POI) from the DD tag. This technique is useful for the regulation of a protein whose free N-terminus is required for its biological function. In this project, we will develop an improved system using short ubiquitin C-terminal sequence to allow more efficient release of a POI from a DD compared to the previous method. The method developed in this project will also be applied for the regulation of endogenous protein expression by combining with genome editing technology.