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Small-molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins

Abstract

The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1G12V-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small-molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models.

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Figure 1: Hydrophobic tagging strategy using the HaloTag fusion protein system.
Figure 2: HyT13 leads to degradation of HaloTag fusion proteins.
Figure 3: Functional validation of HaloTag degradation by HyT13.
Figure 4: Schematic of HyT13 mediated degradation of HaloTag fusion proteins.

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Acknowledgements

We wish to acknowledge financial support from the US National Institutes of Health (R01AI084140) and to thank the members of the Crews lab for critical reading of the manuscript. S. Stricker at the Max Planck Institute for Molecular Genetics kindly provided Mouse Ror2, and R. Weinberg at the Massachusetts Institute of Technology (MIT) contributed Addgene plasmid 9051 from which Hras1G12V was obtained. G. Daley at MIT kindly provided the retroviral pEYK3.1 vector. T.W.C. was the Canadian Institutes of Health Research Jean-François St-Denis Fellow in Cancer Research and a Bisby Fellow. T.B.S. is a recipient of an American Cancer Society fellowship.

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T.K.N., H.S.T., A.R.S. and C.M.C. designed the research. T.K.N., H.S.T., A.R.S., M.J.S., T.W.C., T.B.S. and K.R. performed the experiments. T.K.N., H.S.T., S.A.H. and C.M.C. analyzed the data. T.K.N., H.S.T. and C.M.C. wrote and edited the manuscript.

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Correspondence to Craig M Crews.

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Neklesa, T., Tae, H., Schneekloth, A. et al. Small-molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins. Nat Chem Biol 7, 538–543 (2011). https://doi.org/10.1038/nchembio.597

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