Degradation of HaloTag-fused nuclear proteins using bestatin-HaloTag ligand hybrid molecules.
We have developed a protein knockdown technology using hybrid small molecules designed as conjugates of a ligand for the target protein and a ligand for ubiquitin ligase cellular inhibitor of apoptosis protein 1 (cIAP1). However, this technology has several limitations. Here, we report the development of a novel protein knockdown system to address these limitations. In this system, target proteins are fused with HaloTag to provide a common binding site for a degradation inducer. We designed and synthesized small molecules consisting of alkyl chloride as the HaloTag-binding degradation inducer, which binds to HaloTag, linked to BE04 (2), which binds to cIAP1. Using this system, we successfully knocked down HaloTag-fused cAMP responsive element binding protein 1 (HaloTag-CREB1) and HaloTag-fused c-jun (HaloTag-c-jun), which are ligand-unknown nuclear proteins, in living cells. HaloTag-binding degradation inducers can be synthesized easily, and are expected to be useful as biological tools for pan-degradation of HaloTag-fused proteins.
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. m-ishikawa@iam.u-tokyo.ac.jp.
Shusuke
S
Tomoshige
Mikihiko
M
Naito
Yuichi
Y
Hashimoto
Minoru
M
Ishikawa
D017362:Cyclic AMP Response Element-Binding Protein / Q000037:antagonists & inhibitors*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D007930:Leucine / Q000031:analogs & derivatives* / Q000737:chemistry; D008024:Ligands; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors*; D021381:Protein Transport; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D005456:Fluorescent Dyes; D051034:Inhibitor of Apoptosis Proteins; D008024:Ligands; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D048031:JNK Mitogen-Activated Protein Kinases; D006867:Hydrolases; C046137:haloalkane dehalogenase; D007930:Leucine; C012211:ubenimex