[1S04a] 14. Chemical Biotechnology - Part 2

2:30 PM - 2:45 PM JST(5:30 AM - 5:45 AM UTC)

[1Chemi-08] A Strategy for Intracellular Protein Degradation Control Using a Removable Degron

○LU HAN¹, Yuki Utsugi², Thomas J. Wandless³, Yusaku Miyamae⁴ (1. Doctoral Program in Bioindustrial Sciences, University of Tsukuba (Japan), 2. Master’s/Doctoral Program in Life Science Innovation, University of Tsukuba (Japan), 3. Department of Chemical & Systems Biology, Stanford University (USA), 4. Institute of Life and Environmental Sciences, University of Tsukuba (Japan))

Keywords:

Protein degradation,Degron,Ubiquitin,Deubiquitinating enzymes,Destabilizing domain,Auxin-inducible degron

Protein is the most fundamental biomolecule that play essential roles in living systems, including chemical reactions, molecular transport, and signal transduction. Therefore, technologies that enable precise control and analysis of the expression levels and functions of proteins of interest (POIs) are valuable tools in basic biology and biomedical research. Degron-based systems, which induce protein degradation in response to small molecules, allow rapid, specific, and tunable control of POI abundance. However, these approaches require permanent fusion of degron tags to POIs, which sometimes interferes with their structure and function. To address this limitation, we aimed to develop a removable degron system by introducing a cleavable linker derived from ubiquitin (Ub), allowing conditional release of the degron from the POI. The linker was designed by introducing a point mutation on Gly75 residue of Ub to tune a cleavage rate by cellular deubiquitinating enzymes (DUBs), that are abundantly expressed in cells. All seven Lys residues were substituted to Arg to avoid a self-ubiquitination. We initially tested the Ub linker for combination with destabilizing domain (DD), which induce the expression of POI in a dependent manner upon an addition of stabilizing small molecule ligand, including shield-1. The fusion protein consisting of DD-Ub*-GFP was stably expressed in NIH3T3 cells, mouse-derived fibroblast cells, and tested for cleavage and abundance control by using immunoblotting. In the absence of Shield-1, the fusion protein was rapidly degraded due to unfolding property of DD by cellular proteasome. In contrast, GFP was stabilized and released from the DD by a cleavage of the Ub linker. The system allowed a tunable control on the expression of cleaved GFP upon dose dependent of shiled-1. We also found that the system was applicable to multiple proteins, including mBax, hRPA, H-ras, and Nef. To further evaluate the versatility of this approach, we applied the ubiquitin-based linker to a complement degron system, called auxin-inducible degron 2 (AID2), which induce the protein knockdown in response to 5-phenyl-indole-3-acetic acid (5-Ph-IAA). In HeLa cells expressing AID2–Ub*–POI fusion proteins, cleavage rate of Ub linker was observed greater compared to DD-fusion system. Upon addition of 5-Ph-IAA, AID2-dependent degradation was induced, leading to a marked decrease in POI levels. Quantitative analysis using NanoLuciferase assay demonstrated that POI abundance could be finely tuned in a ligand concentration-dependent manner. Consistent results were obtained with Hras, hRPA, and Src, confirming that both cleavage and degradation can be achieved across different targets. Furthermore, insertion of an HA tag between Ub* and the POI further enhanced DUB-mediated cleavage efficiency. In conclusion, the ubiquitin mutant-based cleavable linker enables a removable degron system that is compatible with both Drug-ON and Drug-OFF platforms. This strategy allows precise control of protein degradation while preserving functional POIs, providing a general and useful tool for protein regulation and functional analysis. This study was partially supported by Grants-in-Aid for Scientific Research (B) [KAKENHI, GRANT no. 25K01910] from the Japan Society for the Promotion of Science.