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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, 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.

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Figure 1: Small molecule–assisted shutoff (SMASh) concept and development.
Figure 2: Proteins can be regulated by SMASh tags at either terminus.
Figure 3: Protein regulation by SMASh-tagging is dose dependent and reversible.
Figure 4: SMASh functions on a variety of proteins.
Figure 5: SMASh functions in S. cerevisiae.
Figure 6: Generation of a drug-controllable 'SMAShable' measles vaccine virus.

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Acknowledgements

We thank M. Billeter (University of Zurich) for p(+)-MeV plasmid, Y. Yanagi (Kyushu University) for Vero-hSLAM cells, M. Takeda (Kyushu University) for the MeV IC-B strain, J. Glenn (Stanford University) for BILN-2061, and A. Gitler, T. Stearns, A. Morrison and J. Skotheim (Stanford University) for yeast plasmids and reagents. We also thank Y. Geng of the Lin laboratory for performing brain dissections, other members of the Lin laboratory for advice, S. Beckwith of the Morrison laboratory for training on yeast procedures and A. Gitler and G. Sherlock (Stanford University) for critical reading of the manuscript. This work was supported by Stanford Graduate Fellowships (H.K.C. and C.L.J.); a US National Science Foundation Graduate Research Fellowship (C.L.J.); NIAID, US National Institutes of Health (NIH) grants 5R01AI071002 and 5R01AI083402 (R.K.P.); NIGMS, NIH EUREKA grant 5R01GM098734 (M.Z.L.); a Burroughs Wellcome Foundation Career Award for Medical Scientists (M.Z.L.); and an Alliance for Cancer Gene Therapy Young Investigator Award (M.Z.L.).

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H.K.C. optimized SMASh, performed mammalian cell, yeast and virus experiments, and wrote the manuscript. C.L.J. performed mammalian cell experiments and contributed to the manuscript. J.Y. optimized SMASh and performed mammalian cell experiments. Y.H. performed mammalian cell experiments. S.A.K. packaged virus. R.K.P. packaged virus and provided advice. R.Y.T. provided advice. M.Z.L. designed SMASh, performed mammalian cell experiments, directed the project and wrote the manuscript.

Corresponding author

Correspondence to Michael Z Lin.

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Chung, H., Jacobs, C., Huo, Y. et al. Tunable and reversible drug control of protein production via a self-excising degron. Nat Chem Biol 11, 713–720 (2015). https://doi.org/10.1038/nchembio.1869

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