Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Sigoillot, F.D. & King, R.W. Vigilance and validation: keys to success in RNAi screening. ACS Chem. Biol. 6, 47–60 (2011).

    CAS  PubMed  Google Scholar 

  2. 2

    Vogel, C. & Marcotte, E.M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat. Rev. Genet. 13, 227–232 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Wu, L. et al. Variation and genetic control of protein abundance in humans. Nature 499, 79–82 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Battle, A. et al. Genomic variation. Impact of regulatory variation from RNA to protein. Science 347, 664–667 (2015).

    CAS  PubMed  Google Scholar 

  5. 5

    Huang, C.J. et al. Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines. Dev. Dyn. 233, 1294–1303 (2005).

    CAS  PubMed  Google Scholar 

  6. 6

    Matsukura, S., Jones, P.A. & Takai, D. Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acids Res. 31, e77 (2003).

    PubMed  PubMed Central  Google Scholar 

  7. 7

    Butko, M.T. et al. Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy. Nat. Neurosci. 15, 1742–1751 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Lin, M.Z., Glenn, J.S. & Tsien, R.Y. A drug-controllable tag for visualizing newly synthesized proteins in cells and whole animals. Proc. Natl. Acad. Sci. USA 105, 7744–7749 (2008).

    CAS  PubMed  Google Scholar 

  9. 9

    Jiang, Y. et al. Discovery of danoprevir (ITMN-191/R7227), a highly selective and potent inhibitor of hepatitis C virus (HCV) NS3/4A protease. J. Med. Chem. 57, 1753–1769 (2014).

    CAS  PubMed  Google Scholar 

  10. 10

    Lamarre, D. et al. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 426, 186–189 (2003).

    CAS  PubMed  Google Scholar 

  11. 11

    McPhee, F. et al. Preclinical profile and characterization of the hepatitis C virus NS3 protease inhibitor asunaprevir (BMS-650032). Antimicrob. Agents Chemother. 56, 5387–5396 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Talwani, R., Heil, E.L., Gilliam, B.L. & Temesgen, Z. Simeprevir: a macrocyclic HCV protease inhibitor. Drugs Today (Barc) 49, 769–779 (2013).

    CAS  Google Scholar 

  13. 13

    Brass, V. et al. Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3–4A complex. Proc. Natl. Acad. Sci. USA 105, 14545–14550 (2008).

    CAS  PubMed  Google Scholar 

  14. 14

    Yao, N., Reichert, P., Taremi, S.S., Prosise, W.W. & Weber, P.C. Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase. Structure 7, 1353–1363 (1999).

    CAS  PubMed  Google Scholar 

  15. 15

    Yuan, L. et al. A rugged and accurate liquid chromatography–tandem mass spectrometry method for the determination of asunaprevir, an NS3 protease inhibitor, in plasma. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 921–922, 81–86 (2013).

    PubMed  Google Scholar 

  16. 16

    Iizuka, R., Yamagishi-Shirasaki, M. & Funatsu, T. Kinetic study of de novo chromophore maturation of fluorescent proteins. Anal. Biochem. 414, 173–178 (2011).

    CAS  PubMed  Google Scholar 

  17. 17

    Mnaimneh, S. et al. Exploration of essential gene functions via titratable promoter alleles. Cell 118, 31–44 (2004).

    CAS  Google Scholar 

  18. 18

    Morawska, M. & Ulrich, H.D. An expanded tool kit for the auxin-inducible degron system in budding yeast. Yeast 30, 341–351 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917–922 (2009).

    CAS  PubMed  Google Scholar 

  20. 20

    Su, L.J. et al. Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models. Dis. Model. Mech. 3, 194–208 (2010).

    CAS  PubMed  Google Scholar 

  21. 21

    Garas, M., Dichtl, B. & Keller, W. The role of the putative 3′ end processing endonuclease Ysh1p in mRNA and snoRNA synthesis. RNA 14, 2671–2684 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Belle, A., Tanay, A., Bitincka, L., Shamir, R. & O'Shea, E.K. Quantification of protein half-lives in the budding yeast proteome. Proc. Natl. Acad. Sci. USA 103, 13004–13009 (2006).

    CAS  PubMed  Google Scholar 

  23. 23

    Miest, T.S. & Cattaneo, R. New viruses for cancer therapy: meeting clinical needs. Nat. Rev. Microbiol. 12, 23–34 (2014).

    CAS  PubMed  Google Scholar 

  24. 24

    Russell, S.J., Peng, K.W. & Bell, J.C. Oncolytic virotherapy. Nat. Biotechnol. 30, 658–670 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Msaouel, P., Opyrchal, M., Domingo Musibay, E. & Galanis, E. Oncolytic measles virus strains as novel anticancer agents. Expert Opin. Biol. Ther. 13, 483–502 (2013).

    CAS  PubMed  Google Scholar 

  26. 26

    Rima, B.K. & Duprex, W.P. The measles virus replication cycle. Curr. Top. Microbiol. Immunol. 329, 77–102 (2009).

    CAS  PubMed  Google Scholar 

  27. 27

    Zuniga, A. et al. Attenuated measles virus as a vaccine vector. Vaccine 25, 2974–2983 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Lampson, M.A. & Kapoor, T.M. Targeting protein stability with a small molecule. Cell 126, 827–829 (2006).

    CAS  PubMed  Google Scholar 

  29. 29

    Banaszynski, L.A., Chen, L.-C., Maynard-Smith, L.A., Ooi, A.G.L. & Wandless, T.J. A rapid, reversible and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Iwamoto, M., Bjorklund, T., Lundberg, C., Kirik, D. & Wandless, T.J. A generalchemical method to regulate protein stability in the mammalian central nervous system. Chem. Biol. 17, 981–988 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Stankunas, K. et al. Conditional protein alleles using knock-in mice and a chemical inducer of dimerization. Mol. Cell 12, 1615–1624 (2003).

    CAS  Google Scholar 

  32. 32

    Bonger, K.M., Chen, L.C., Liu, C.W. & Wandless, T.J. Small-molecule displacement of a cryptic degron causes conditional protein degradation. Nat. Chem. Biol. 7, 531–537 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Tae, H.S. et al. Identification of hydrophobic tags for the degradation of stabilized proteins. ChemBioChem 13, 538–541 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Andresen, M., Schmitz-Salue, R. & Jakobs, S. Short tetracysteine tags to β-tubulin demonstrate the significance of small labels for live cell imaging. Mol. Biol. Cell 15, 5616–5622 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Chen, M., Cortay, J.C. & Gerlier, D. Measles virus protein interactions in yeast: new findings and caveats. Virus Res. 98, 123–129 (2003).

    CAS  PubMed  Google Scholar 

  36. 36

    Shu, Y. et al. Plasticity in structural and functional interactions between the phosphoprotein and nucleoprotein of measles virus. J. Biol. Chem. 287, 11951–11967 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Devaux, P. & Cattaneo, R. Measles virus phosphoprotein gene products: conformational flexibility of the P/V protein amino-terminal domain and C protein infectivity factor function. J. Virol. 78, 11632–11640 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Huh, W.K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Pratt, M.R., Schwartz, E.C. & Muir, T.W. Small-molecule–mediated rescue of protein function by an inducible proteolytic shunt. Proc. Natl. Acad. Sci. USA 104, 11209–11214 (2007).

    CAS  PubMed  Google Scholar 

  40. 40

    Lin, Y.H. & Pratt, M.R. A dual small-molecule rheostat for precise control of protein concentration in mammalian cells. ChemBioChem 15, 805–809 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Cong, F., Zhang, J., Pao, W., Zhou, P. & Varmus, H. A protein knockdown strategy to study the function of β-catenin in tumorigenesis. BMC Mol. Biol. 4, 10 (2003).

    PubMed  PubMed Central  Google Scholar 

  42. 42

    Sakamoto, K.M. et al. Protacs: chimeric molecules that target proteins to the Skp1–Cullin–F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 98, 8554–8559 (2001).

    CAS  PubMed  Google Scholar 

  43. 43

    Limenitakis, J. & Soldati-Favre, D. Functional genetics in Apicomplexa: potentials and limits. FEBS Lett. 585, 1579–1588 (2011).

    CAS  PubMed  Google Scholar 

  44. 44

    Mumberg, D., Muller, R. & Funk, M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122 (1995).

    CAS  Google Scholar 

  45. 45

    Shimobayashi, M. & Hall, M.N. Making new contacts: the mTOR network in metabolism and signaling crosstalk. Nat. Rev. Mol. Cell Biol. 15, 155–162 (2014).

    CAS  PubMed  Google Scholar 

  46. 46

    Zhou, P. Determining protein half-lives. Methods Mol. Biol. 284, 67–77 (2004).

    CAS  PubMed  Google Scholar 

  47. 47

    Shoulders, M.D., Ryno, L.M., Cooley, C.B., Kelly, J.W. & Wiseman, R.L. Broadly applicable methodology for the rapid and dosable small molecule–mediated regulation of transcription factors in human cells. J. Am. Chem. Soc. 135, 8129–8132 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Cattaneo, R., Miest, T., Shashkova, E.V. & Barry, M.A. Reprogrammed viruses as cancer therapeutics: targeted, armed and shielded. Nat. Rev. Microbiol. 6, 529–540 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Meng, X. et al. Enhanced antitumor effects of an engineered measles virus Edmonston strain expressing the wild-type N, P, L genes on human renal cell carcinoma. Mol. Ther. 18, 544–551 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Chen, N. et al. Poxvirus interleukin-4 expression overcomes inherent resistance and vaccine-induced immunity: pathogenesis, prophylaxis and antiviral therapy. Virology 409, 328–337 (2011).

    CAS  PubMed  Google Scholar 

  51. 51

    Thomas, B.J. & Rothstein, R. Elevated recombination rates in transcriptionally active DNA. Cell 56, 619–630 (1989).

    CAS  Google Scholar 

  52. 52

    Brindley, M.A. et al. A stabilized headless measles virus attachment protein stalk efficiently triggers membrane fusion. J. Virol. 87, 11693–11703 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Buchholz, U.J., Finke, S. & Conzelmann, K.K. Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J. Virol. 73, 251–259 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Krumm, S.A., Takeda, M. & Plemper, R.K. The measles virus nucleocapsid protein tail domain is dispensable for viral polymerase recruitment and activity. J. Biol. Chem. 288, 29943–29953 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Ono, N. et al. Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor. J. Virol. 75, 4399–4401 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Plemper, R.K., Hammond, A.L., Gerlier, D., Fielding, A.K. & Cattaneo, R. Strength of envelope protein interaction modulates cytopathicity of measles virus. J. Virol. 76, 5051–5061 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


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.).

Author information




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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–9. (PDF 7792 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

Further reading


Quick links