Tunable and reversible drug control of protein production via a self-excising degron

Abstract

An effective method for direct chemical control over the production of specific proteins would be widely useful. We describe Small Molecule-Assisted Shutoff (SMASh), a technique in which proteins are fused to a degron that removes itself in the absence of drug, leaving untagged protein. Clinically tested HCV protease inhibitors can then block degron removal, inducing rapid degradation of subsequently synthesized protein copies. SMASh allows reversible and dose-dependent shutoff of various proteins in multiple mammalian cell types and in yeast. We also used SMASh to confer drug responsiveness onto a RNA virus for which no licensed inhibitors exist. As SMASh does not require permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. Furthermore, as SMASh only involves a single genetic modification and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts.

A small-molecule SMASh hit

“…The system reported in this issue by Chung et al 1 is minimally disruptive, effective and inducible. The protein of interest is tagged with the hepatitis C virus protease NS3 as well as a destabilizing degron (Fig.…”

“…To accurately decipher how genes function within the cell, tight control of their intracellular abundance is essential. In this issue, Chung et al 1 report the SMASh system, in which gene products are fused to a drug-sensitive viral protease and a protein destabilization element (degron), which allows for a simple way to turn off expression of the protein of interest 1 . This approach, which is simple, straightforward and conducive to high-throughput technology, has clear applications as a research tool and may be potentially useful for therapeutic systems.…”

“…In their report, Chung et al 1 demonstrate that the SMASh system requires minimal genetic modification. The degron-protease tag works on multiple target genes and is equally effective whether attached to the N or C terminus of the protein of interest.…”

“…Although the SMASh system cannot be used for direct targeting of endogenous proteins, as certain chimeric E3 ligases can, the SMASh tag can be added to endogenous genes 7,8 . A recent report used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene-editing system to add inducible degrons to the TCOF1 locus in human cells, and Chung et al 1 demonstrate in their report that SMASh tag modification of endogenous yeast genes ( YSH1 and SEC14 ) is feasible 1,9 .…”

A Novel Destabilizing Domain Based on a Small-Molecule Dependent Fluorophore

“…More recently, Lin and coworkers engineered a protein domain that comprises a degron fused to an HCV protease and its cognate recognition site. 8 In the absence of an HCV protease inhibitor, the protease excises itself and the degron from the fusion protein, thereby releasing the untagged protein. The addition of the inhibitor prevents the protease from engaging its cleavage site; thus, the fusion protein remains tagged with a degron and is rapidly degraded.…”