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ACS Chem Biol. 2013 Oct 18;8(10):2293-2300. doi: 10.1021/cb400569k. Epub 2013 Aug 26.

A bidirectional system for the dynamic small molecule control of intracellular fusion proteins.

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Department of Molecular Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, CT 06511, USA.
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA.
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School-UMDNJ, Piscataway, NJ 08854, USA.
Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511, USA.
Department of Pharmacology, Yale University, 333 Cedar Street, New Haven, CT 06511, USA.
Contributed equally


Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function. Recently, we developed a method to degrade proteins fused to a mutant dehalogenase (HaloTag2) using small molecule hydrophobic tags (HyTs). Here, we introduce a complementary method to stabilize the same HaloTag2 fusion proteins, resulting in a unified system allowing bidirectional control of cellular protein levels in a temporal and dose-dependent manner. From a small molecule screen, we identified N-(3,5-dichloro-2-ethoxybenzyl)-2H-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1) that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2 ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS system in probing the physiological role of therapeutic targets by modulating HaloTag2-fused oncogenic H-Ras, which resulted in either the cessation (HyT) or acceleration (HALTS) of cellular transformation. In sum, we present a general platform to study protein function, whereby any protein of interest fused to HaloTag2 can be either degraded 10-fold or stabilized 5-fold using two corresponding compounds.

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