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

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• 作者：Taavi K. Neklesa ; Devin J. Noblin ; Alexander Kuzin ; Scott Lew ; Jayaraman Seetharaman ; Thomas B. Acton ; Gregory Kornhaber ; Rong Xiao ; Gaetano T. Montelione ; Liang Tong ; Craig M. Crews
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：October 18, 2013
• 年：2013
• 卷：8
• 期：10
• 页码：2293-2300
• 全文大小：454K
• 年卷期：v.8,no.10(October 18, 2013)
• ISSN：1554-8937

文摘

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.