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Oxysterols are allosteric activators of the oncoprotein Smoothened

Nature Chemical Biology volume 8, pages 211–220 (2012)Cite this article

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Abstract

Oxysterols are a class of endogenous signaling molecules that can activate the Hedgehog pathway, which has critical roles in development, regeneration and cancer. However, it has been unclear how oxysterols influence Hedgehog signaling, including whether their effects are mediated through a protein target or indirectly through effects on membrane properties. To answer this question, we synthesized the enantiomer and an epimer of the most potent oxysterol, 20(S)-hydroxycholesterol. Using these molecules, we show that the effects of oxysterols on Hedgehog signaling are exquisitely stereoselective, consistent with the hypothesis that they function through a specific protein target. We present several lines of evidence that this protein target is the seven-pass transmembrane protein Smoothened, a major drug target in oncology. Our work suggests that these enigmatic sterols, which have multiple effects on cell physiology, may act as ligands for signaling receptors and provides a generally applicable framework for probing sterol signaling mechanisms.

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Figure 1: Activation of Hedgehog signaling by oxysterols is regioselective.
Figure 2: Activation of Hedgehog signaling by nat-20(S)-OHC is stereoselective.
Figure 3: Distinct pharmacological interactions of Smo inhibitors with nat-20(S)-OHC.
Figure 4: Synergistic activation of Hedgehog signaling by nat-20(S)-OHC and SAG.
Figure 5: Smo binds a nat-20(S)-OHC analog immobilized on beads.
Figure 6: A model for the allosteric regulation of Smo by small molecules.

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Acknowledgements

We thank members of the Rohatgi lab for helpful discussions, G. Luchetti for help with ligand affinity chromatography, P. Niewiadomski and A. Lebensohn for critical reading of the manuscript, E. Lee (Vanderbilt University) for Wnt-L cells, K. Mykytyn (Ohio State University) for the SSTR3-GFP and HTR6-GFP constructs, A. Sweet-Cordero for use of a Li-Cor Odyssey imager and M. Scott for support and use of a confocal microscope. MS analysis was conducted at the NIH – National Center for Research Resources MS facility at Washington University, supported by the NIH (RR00954, DK020579, DK056341). This work was supported by a Pew Scholar Award and an Innovation Research Grant from the Stand Up to Cancer – American Association for Cancer Research Foundation to R.R., by NIH grants to D.F.C. (GM47969 and HL67773) and P.H.S. (HL67773), and by NIH training grants to S.N. (5 T32 GM007276) and L.K.M. (5 T32 HL007275).

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Authors and Affiliations

  1. Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA

    Sigrid Nachtergaele

  2. Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, USA

    Laurel K Mydock, Kathiresan Krishnan & Douglas F Covey

  3. Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, USA

    Jayan Rammohan & Paul H Schlesinger

  4. Department of Medicine, Stanford University School of Medicine, Stanford, California, USA

    Rajat Rohatgi

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Contributions

L.K.M., K.K. and D.F.C. designed and synthesized the oxysterol analogs. S.N. performed cellular experiments with oxysterols. J.R. and P.H.S. designed and performed vesicle expansion experiments. All authors analyzed the data and contributed to the manuscript. S.N. and R.R. wrote the paper with input from L.K.M., D.F.C., J.R. and P.H.S.

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Correspondence to Douglas F Covey or Rajat Rohatgi.

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Nachtergaele, S., Mydock, L., Krishnan, K. et al. Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol 8, 211–220 (2012). https://doi.org/10.1038/nchembio.765

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