A Bidirectional System for the Dynamic Small Molecule Control of Intracellular Fusion Proteins

作者：Taavi K. Neklesa, Devin J. Noblin, A. Kuzin, Scott Lew, J. Seetharaman, Thomas Acton, Gregory J. Kornhaber, Rong Xiao, Gaetano Thomas Montelione, Liang Tong, Craig M. Crews · 发表于：ACS Chemical Biology · 年份：2013 · DOI：10.1021/cb400569k · 被引用次数：52 · 研究领域：Viral Infectious Diseases and Gene Expression in Insects、Protein Degradation and Inhibitors、Virus-based gene therapy research

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.