A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment
Gang Chen,
Yu Kong,
You Li,
Ailing Huang,
Chunyu Wang,
Shanshan Zhou,
Zhenlin Yang,
Yanling Wu,
Jianke Ren,
Tianlei Ying
Affiliations
Gang Chen
MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
Yu Kong
MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
You Li
MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
Ailing Huang
MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
Chunyu Wang
MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
Shanshan Zhou
MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
Zhenlin Yang
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China
Yanling Wu
MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
Jianke Ren
CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Tianlei Ying
MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin–proteasome and autophagy–lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.
