A Bidirectional System for the Dynamic Small Molecule
Control of Intracellular Fusion Proteins
- Publication date
- Publisher
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 <i>N</i>-(3,5-dichloro-2-ethoxybenzyl)-2<i>H</i>-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