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Volume 32, No 3, Mar 2022

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 32 Issue 3, March 2022: 288-301

ORIGINAL ARTICLES

Cholesterylation of Smoothened is a calcium-accelerated autoreaction involving an intramolecular ester intermediate

Ao Hu1,† , Jing-Zan Zhang2,† , Jie Wang2 , Chen-Chen Li2 , Meng Yuan1 , Gang Deng1 , Zi-Cun Lin1 , Zhi-Ping Qiu1 , Hu-Yue Liu1 , Xian-Wei Wang2,3 , Peng-Cheng Wei1 , Xiao He2,4 , Xiaolu Zhao1,* , Wen-Wei Qiu2,* , Bao-Liang Song1,*

1The Institute for Advanced Studies, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, TaiKang Medical School, Wuhan University, Wuhan, Hubei, China
2Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai, China
3College of Science, Zhejiang University of Technology, Hangzhou, Zhejiang, China
4NYU-ECNU Center for Computational Chemistry at NYU Shanghai, Shanghai, China
†These authors contributed equally: Ao Hu, Jing-Zan Zhang
Correspondence: Xiaolu Zhao(zhaoxiaolu@whu.edu.cn)Wen-Wei Qiu(wwqiu@chem.ecnu.edu.cn)Bao-Liang Song(blsong@whu.edu.cn)

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.


https://doi.org/10.1038/s41422-022-00622-0

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