Hedgehog Signal Transduction
Several key questions regarding the biogenesis, tissue distribution, and transduction of the hedgehog signal are under active investigation in our lab:
The Hedgehog morphogen activates the pathway by binding to the 12TM protein Patched, thus relieving suppression of the GPCR family protein, Smoothened; this in turn leads to activation of the Gli zinc finger transcriptional effectors. Although isolation of Patched genes and characterization of ShhN binding to Patched1-expressing cells was accomplished many years ago, Patched regulation of Smoothened remains the central mystery of Hedgehog signal transduction. How does Patched inhibit Smoothened activation, and how does binding to Hedgehog inactivate Patched to relieve this inhibition? We showed previously that Patched acts sub-stoichiometrically to regulate Smoothened and that conserved sequence motifs shared between Patched and members of the prokaryotic resistance-nodulation-division transmembrane transporter family are required for its action. In addition, Smoothened binds to and is regulated by natural as well as synthetic lipidic molecules, and we showed many years ago that cholesterol is required for Smoothened activation. We have proposed that Patched acts as a transporter to control access of Smoothened to an endogenous lipid that regulates it.
Whereas Patched–Smoothened regulation has traditionally been studied through downstream assays of Hedgehog-dependent transcriptional activity, we recently developed rapid, direct functional assays to dissect this regulation in simplified cell-based and in vitro systems. We have found that Patched1–Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. Furthermore, Patched1 regulation of Smoothened can occur in the absence of the Smoothened extracellular domain. We also find that cholesterol, a major membrane lipid, constitutively activates purified Smoothened by engaging its 7TM region. Patched1 activity depends on extracellular Na+, suggesting that transmembrane Na+ gradients, universal among metazoans, might power Patched1 transporterlike activity in Smoothened regulation. We continue to investigate the biochemical and structural basis of Smoothened activation, and the structure of Patched and its biochemical mechanism.
We have shown that the active Hedgehog signal is a cholesterol- and palmitate-modified amino-terminal fragment of the secreted precursor, generated by an autoprocessing reaction (concerted peptide bond cleavage/cholesterol addition) and an acyl transfer reaction (N-terminal palmitoylation). How is it possible that dually lipid-modified Hedgehog, expected to remain membrane-tethered, moves through the extracellular milieu to exert a direct signaling effect across many cell diameters? We have shown that the lipidated Hedgehog fragment, designated ShhNp (Sonic hedgehog N-terminal fragment, processed), requires the proteins Dispatched and You/Scube2 for release. We continue to investigate the molecular mechanism of Hedgehog packaging and release, and the form of the diffusible, packaged Hedgehog morphogen.
Select Reviews and Publications:
Wang, Q., Asarnow, D.E., Ding, K., Mann, R.K., Hatakeyama, J., Zhang, Y., Ma, Y., Cheng, Y., Beachy, P.A. Dispatched uses Na+ flux to power release of lipid-modified Hedgehog. Nature 599(7884): 320-324 (2021). Pubmed
Zhang, Y., Lu, W.J., Bulkley, D.P., Liang, J., Ralko, A., Han, S., Roberts, K.J., Li, A., Cho, W., Cheng, Y., Manglik, A., Beachy, P.A. Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit. Proc Natl Acad Sci U S A.117(46):28838-28846 (2020). Pubmed
Deshpande, I., Liang J., Hedeen, D., Roberts, K.J., Zhang, Y., Ha, B., Latorraca, N.R., Faust, B., Dror R.O., Beachy, P.A., Myers, B.R., Manglik, A. Smoothened stimulation by membrane sterols drives Hedgehog pathway activity. Nature 571(7764): 284-288 (2019). Pubmed
Zhang, Y., Bulkley, D., Xin, Y., Roberts, K., Asarnow, D., Sharma, A., Myers, B., Cho, W., Cheng, Y., Beachy, P.A. Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched. Cell, 175(5):1352-1364 (2018). Pubmed
Myers, B.R., Neahring, L.., Zhang, Y., Roberts, K.J., Beachy, P.A. Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium. PNAS 114(52):E11141-E11150 (2017). Pubmed
Kim, J., Hsia, E., Brigui, A., Plessis, A., Beachy, P.A.* Zheng, X.* The role of ciliary trafficking in Hedgehog receptor signalings. Science Signaling 8:379 (2015). Pubmed
Creanga, A., Glenn, T.D., Mann, R.K., Saunders, A.M., Talbot, W.S., Beachy, P.A. Scube/You activity mediates release of dually lipid-modified Hedgheog signal in soluble form. Genes & Dev. 26(12):1312-25 (2012). Pubmed
Beachy, P.A., Hymowitz, S.G., Lazarus, R.A., Leahy, D.J., Siebold, C. Interactions between Hedgehog proteins and their binding partners come into view. Genes & Dev, 24(18):2001-12 (2010). Pubmed
Mann, R.K. and Beachy, P.A. Novel lipid modifications of secreted protein signals. Ann. Rev. Biochem. 73:891-923 (2004). Pubmed
Cooper, M.K., Wassif, C.A., Krakowiak, P.A., Taipale, J., Gong, R., Kelley, R.I., Porter, F.D., Beachy, P.A. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat Genet 33(4):508-13 (2003). Pubmed
Taipale, J., Cooper, M.K., Maiti, T., Beachy, P.A. Patched acts catalytically to suppress the activity of Smoothened. Nature 418:892-896 (2002). Pubmed
Chen, J.K., Taipale, J., Cooper, M.K., Beachy, P.A. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes & Dev. 16: 2743-2748 (2002). Pubmed
Ma, Y., Erkner, A., Gong, R., Yao, S., Taipale, J., Basler, K., Beachy, P.A. Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of Dispatched. Cell 111(1):63-75 (2002). Pubmed
Chamoun, Z., Mann, R.K., Nellen, D., von Kessler, D.P., Bellotto, M., Beachy, P.A., Basler, K. Skinny Hedgehog, an acyltransferase required for palmitoylation and activity of the Hedgehog signal. Science 293(5537):2080-4 (2001). Pubmed
Fuse, N., Maiti, T., Wang, B., Porter, J.A., Hall, T.M., Leahy, D.J., Beachy, P.A. Sonic Hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for Patched. Proc Natl Acad Sci 96(20):10992-9 (1999). Pubmed
Porter, J.A., Young, K.E., Beachy, P.A. Cholesterol modification of Hedgehog signaling proteins in animal development. Science 274:255-259 (1996). Pubmed
Lee, J.J., von Kessler, D.P., Parks, S., and Beachy, P.A. Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene Hedgehog. Cell 71:33-50 (1992). Pubmed