2. Academic Validation
  3. A bidirectional system for the dynamic small molecule control of intracellular fusion proteins

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

  • ACS Chem Biol. 2013 Oct 18;8(10):2293-2300. doi: 10.1021/cb400569k.
Taavi K Neklesa # 1,  Devin J Noblin # 1,  Alexander P Kuzin 2,  Scott Lew 2,  Jayaraman Seetharaman 2,  Thomas B Acton 3,  Gregory J Kornhaber 3,  Rong Xiao 3,  Gaetano Thomas Montelione 3 4,  Liang Tong 2,  Craig M Crews 1 5 6
Affiliations

Affiliations

  • 1 Department of Molecular Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, CT 06511, USA.
  • 2 Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA.
  • 3 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.
  • 4 Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School-UMDNJ, Piscataway, NJ 08854, USA.
  • 5 Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511, USA.
  • 6 Department of Pharmacology, Yale University, 333 Cedar Street, New Haven, CT 06511, USA.
  • # Contributed equally.
PMID: 23978068 DOI: 10.1021/cb400569k
Abstract

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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