Volume 66, Issue 1, Pages e10 (April 2017)

Volume 66, Issue 1, Pages 154-162.e10 (April 2017)
Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling Xu Xiao, Jing-Jie Tang, Chao Peng, Yan Wang, Lin Fu, Zhi-Ping Qiu, Yue Xiong, Lian-Fang Yang, Hai-Wei Cui, Xiao-Long He, Lei Yin, Wei Qi, Catherine C.L. Wong, Yun Zhao, Bo-Liang Li, Wen-Wei Qiu, Bao-Liang Song Molecular Cell Volume 66, Issue 1, Pages e10 (April 2017) DOI: /j.molcel Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 66, 154-162.e10DOI: (10.1016/j.molcel.2017.02.015)
Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 SMO Is Covalently Modified by the Cholesterol Probe (CP)
(A) Schematic representation of the procedure to detect and identify CP-modified protein(s). (B) HEK293T cells expressing precursor Shh were subjected to experiments depicted in Figure 1A. Each fraction was examined by western blotting. Pre-Shh, precursor Shh; N-Shh-cholesterol/CP, N terminus of Shh covalently modified by cholesterol or CP; N-Shh-CP-biotin, N terminus of Shh covalently modified by CP that was further linked to biotin. (C) SMO was modified by CP in a concentration-dependent manner. HEK293T cells were transfected with SMO expression plasmid, incubated in cholesterol-depleting medium supplemented with increasing concentrations of CP for 16 hr, and then subjected to procedures as shown in Figure 1A. The pellet (Fraction 4) and input (Fraction 2) were analyzed by western blotting. SMO-CP-Biotin, SMO covalently linked with CP and biotin alkyne. (D) Endogenous SMO was modified by CP. HEK293T cells were harvested and analyzed as depicted in Figure 1A. (E) CP-modified SMO was precipitated in both nondenature and denature conditions. The experiment was performed as depicted in Figure 1A. After click chemistry reaction, the samples were incubated in nondenature buffer or denature buffer followed by precipitation with neutravidin beads. (F) SMO modified by CP was outcompeted by cholesterol. HEK293T cells were transfected with SMO expression plasmid, incubated in cholesterol-depleting medium supplemented with 2 μg/mL CP plus indicated concentrations of cholesterol for 16 hr, and then subjected to procedures as shown in Figure 1A. See also Figures S1, S2, and S3. Molecular Cell , e10DOI: ( /j.molcel ) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 The D95 Residue Is the Cholesterol Modification Site of Human SMO (A) Tandem Mass spectrometry (MS/MS) spectrum showing that SMO was modified on D95 by cholesterol. HEK293T cells were transfected with SMO-3 × FLAG expression plasmid, lysed, and immunoprecipitated with anti-FLAG beads. After elution with 3 × FLAG peptide, the sample was analyzed by MS/MS. (B) CP modification of SMO variants. The experiment procedure used here was depicted in Figure 1A. (C) Model of the related amino acids around the cholesterol-binding groove. The model is based on the crystal structure of the human SMO (PDB entry 5l7d). Residues in red were essential for cholesterol binding to SMO-CRD. Residues in brown partially affected cholesterol binding, and those in black had no effect on cholesterol binding. (D) CP modification of SMO variants. Residues in red were essential for cholesterol binding to CRD. Residues in brown partially affected cholesterol binding, and those in black had no effect on cholesterol binding. See also Figure S3. Molecular Cell , e10DOI: ( /j.molcel ) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 The Cholesterylation of SMO Is Regulated by Hh Signaling
(A) Ptch1 inhibited CP modification of SMO. After cotransfection, HEK293T cells were subjected to experiment procedures as depicted in Figure 1A. (B) CP modification of SMO was inhibited by WT Ptch1, but not by loss-of-function mutant Ptch1(G472R). (C) Knockdown of Ptch1 enhanced CP modification of endogenous SMO. qPCR showed the knockdown efficiency at the bottom. Data are expressed as mean ± SD. (D) Coexpression of Shh increased CP modification of SMO. (E) The N-Shh protein increased CP modification of endogenous SMO. Molecular Cell , e10DOI: ( /j.molcel ) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 The D95 Residue Is Essential for SMO Ciliary Localization, Activity, and Function (A) The ciliary localization of SMO variants (WT, D95N, D97N, and Y130F) in NIH 3T3 cells treated with or without N-Shh. (B) Quantification of SMO-positive cilia in (A). n = 80–100; statistical analyses, two-way ANOVA; data are expressed as mean ± SD. ∗∗∗p < 0.001, compared with SMO(WT) without N-Shh stimulation; ns, not statistically significant compared with SMO(WT) without N-Shh stimulation; ##p < 0.01, ###p < 0.001, compared with the corresponding SMO variant without N-Shh stimulation; NS, not statistically significant compared with the corresponding SMO variant without N-Shh stimulation. (C) Quantification of fluorescent intensity of ciliary SMO. n = 30–40; statistical analyses, two-way ANOVA; data are expressed as mean ± SD. ∗∗∗p < 0.001, compared with SMO(WT) without N-Shh stimulation; ns, not statistically significant compared with SMO(WT) without N-Shh stimulation; ##p < 0.01, ###p < 0.001, compared with the corresponding SMO variant without N-Shh stimulation; NS, not statistically significant compared with the corresponding SMO variant without N-Shh stimulation. (D) The Gli-Luc reporter assay. Statistical analyses, two-way ANOVA; data are expressed as mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001, compared with SMO(WT) without N-Shh stimulation; ns, not statistically significant compared with SMO(WT) without N-Shh stimulation; #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the corresponding SMO variant without N-Shh stimulation; NS, not statistically significant compared with the corresponding SMO variant without N-Shh stimulation. (E) External morphology of the 9.5 days post coitum (DPC) embryos. The D99 of mouse SMO is equivalent to D95 of human SMO. (F) Cross-sections of the 9.5 DPC embryos. bc, bulbus cordis; v, ventricle. (G) Immunofluorescent staining of the neural tube sections of 9.5 DPC embryos for Olig2 and Nkx6.1. (H) The expression levels of hedgehog-pathway-related genes in the whole embryos. Statistical analyses, one-way ANOVA; data are expressed as mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001, compared with WT mice; ns, not statistically significant compared with WT mice. See also Figure S4. Molecular Cell , e10DOI: ( /j.molcel ) Copyright © 2017 Elsevier Inc. Terms and Conditions

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Volume 66, Issue 1, Pages e10 (April 2017)

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