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

ARTICLE

PUBLISHED ONLINE: 27 JULY 2015 | http://www.nature.com/doifinder/10.1038/nchembio.1869

Web End =DOI: 10.1038/NCHEMBIO.1869

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

Hokyung K Chung1, Conor L Jacobs1, Yunwen Huo2, Jin Yang3, Stefanie A Krumm4, Richard K Plemper4,5, Roger Y Tsien3,6,7 & Michael Z Lin2,8*

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

npg 201 5 Nature America, Inc. All rights reserved.

Technology for rapidly shutting off the production of specific proteins in eukaryotes would be widely useful in research and in gene and cell therapies, but a simple and effective method

has yet to be developed. Controlling protein production through repression of transcription is slow in onset because previously transcribed mRNA molecules continue to produce proteins. RNA interference (RNAi) induces mRNA destruction, but RNAi is often only partially effective and can exhibit both sequence-independent and sequence-dependent off-target effects1. Furthermore, mRNA and protein abundance are not always correlated as a result of the translational regulation of specific mRNAs24. Lastly, both transcriptional repression and RNAi take days to reverse5,6.

To address these limitations, we wished to devise a method for chemical regulation of protein expression at the post-translational level. An ideal method would feature (i) genetic specification of the target protein, (ii) a single genetic modification for simplicity, (iii) minimal modification of the expressed protein, (iv) generaliz-ability to many proteins and cell types...