Abstract
Hedgehog (Hh) is a morphogen that binds to its receptor Patched 1 and activates Smoothened (SMO), thereby governing embryonic development and postnatal tissue homeostasis. Cholesterol can bind and covalently conjugate to the luminal cysteine-rich domain (CRD) of human SMO at the D95 residue (D99 in mouse). The reaction mechanism and biological function of SMO cholesterylation have not been elucidated. Here, we show that the SMO-CRD undergoes auto-cholesterylation which is boosted by calcium and involves an intramolecular ester intermediate. In cells, Hh stimulation elevates local calcium concentration in the SMO-localized endosomes through store-operated calcium entry. In addition, we identify the signaling-incompetent SMO D95E mutation, and the D95E mutant SMO can bind cholesterol but cannot be modified or activated by cholesterol. The homozygous SmoD99E/D99E knockin mice are embryonic lethal with severe developmental delay, demonstrating that cholesterylation of CRD is required for full-length SMO activation. Our work reveals the unique autocatalytic mechanism of SMO cholesterylation and an unprecedented role of calcium in Hh signaling.
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Acknowledgements
We thank Ms. Dan Liang for technical assistance, Dr. Wei Qi (ShanghaiTech University) for revising the manuscript. We appreciate the helpful discussion with Drs. Guo-Yin Yin (Wuhan University) and Yun Zhao (Shanghai Institutes for Biological Sciences). This work was supported by grants from the National Natural Science Foundation of China (22077035, 91753204, 31690102, 91957103), Ministry of Science and Technology of China (2018YFA0800700, 2019YFA0802701), and Fountain-Valley Life Sciences Fund of University of Chinese Academy of Sciences Education Foundation. B.-L.S. acknowledges the support from the Tencent Foundation through the XPLORER PRIZE.
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B.-L.S. conceived the project. J.-Z.Z., C.-C.L, J.W. and W.-W.Q. designed and synthesized cholesterol analogs. A.H. and B.-L.S. designed the experiments. A.H., Z.-C.L., Z.-P.Q., and H.-Y.L. performed the experiments. P.-C.W. performed the structural remodeling. M.Y., G.D. and X.Z. did MS/MS. X.-W.W. and X.H. performed quantum mechanical calculations. A.H. and B.-L.S. analyzed the data. A.H., J.W., W.-W.Q. and B.-L.S. wrote the paper with input from others.
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Hu, A., Zhang, JZ., Wang, J. et al. Cholesterylation of Smoothened is a calcium-accelerated autoreaction involving an intramolecular ester intermediate. Cell Res 32, 288–301 (2022). https://doi.org/10.1038/s41422-022-00622-0
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DOI: https://doi.org/10.1038/s41422-022-00622-0
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