An IMiD-induced SALL4 degron system for selective degradation of target proteins

Satoshi Yamanaka,Saya Matsuoka,Tatsuya Sawasaki,Hisayo Nishida-Fukuda,Norio Shibata,Yuki Shoya

doi:10.1038/s42003-020-01240-5

Abstract

Regulating the amount of proteins in living cells is a powerful approach for understanding the functions of the proteins. Immunomodulatory drugs (IMiDs) induce the degradation of neosubstrates by interacting with celebron (CRBN) in the cullin E3 ubiquitin ligase complex (CRL4CRBN). Here, we developed the IMiD-dependent Sal-like protein 4 (SALL4) degron (S4D) system for chemical protein knockdown. In transient assays, an N- or C-terminal S4D tag induced the degradation of proteins localized to various subcellular compartments, including the plasma membrane. The activity of luciferase-S4D was reduced by 90% within 3 h of IMiD treatment. IMiD treatment reduced the expression of endogenous S4D-fused RelA and IκBα in knock-in (KI) experiments. Interestingly, the IκBα knockdown suggested that there may be another, unknown mechanism for RelA translocation to the nucleus. Furthermore, 5-hydroxythalidomide as a thalidomide metabolite specifically degradated S4D-tagged protein. These results indicate that the S4D system is a useful tool for cellular biology.

Highlights

Regulating the amount of proteins in living cells is a powerful approach for understanding the functions of the proteins
Because the m2 sequence is shorter than m1, m2 was selected as the Sal-like protein 4 (SALL4) degron tag and named S4D
We investigated whether IMiD-dependent protein degradation of AGIA-Venus-S4D is induced by endogenous CRBN because exogenous FLAG-CRBN was increased by IMiD treatment

Summary

Introduction

Regulating the amount of proteins in living cells is a powerful approach for understanding the functions of the proteins. Many systems, including the auxin-inducible degron (AID) system, combine genetic and chemical strategies to achieve protein knockdown[2,3,4]. These systems combine the specificity of genetic approaches with the ease of use of small molecule compounds[1]. Researchers need to consider the effects of the additional tag on the localization or activity of the protein of interest These systems offer some improvements for the utilization in cell biology. IKZF3 and SALL4 interact with the CRBN-IMiD complex through a single C2H2 zinc finger domain (ZNF)[8,9,10]. Its use was still limited to the C-terminus of the protein of interest and overexpressed tagged proteins, not endogenous proteins[11]

Methods

Results

Conclusion