The Hedgehog (Hh) signaling pathway is critical for embryonic development and tissue renewal. The G protein-coupled receptor (GPCR)-like protein Smoothened (SMO) is the central signal transducer in the Hh pathway. Cholesterol binds and then covalently links to the D95 residue of cysteine-rich domain (CRD) of human SMO. The cholesterylation of CRD is critical for SMO activation. SMO cholesterylation is a Ca 2+-boosted autoreaction that requires the formation of an ester bond between the side chains of D95 and Y130 as an intermediate. It is unknown whether other residues of SMO are involved in the esterification between D95 and cholesterol. In this study, we find that the SMO-CRD(27-192) can undergo cholesterylation. In addition to D95 and Y130, the residues critical for cholesterol modification include Y85, T88, T90, W109, W119, K133, E160 and F166. T88, W109, W119 and F166 also seem to be involved in protein folding. Notably, we find that Y85 and K133 form a cation-π interaction whose disruption abolishes cholesterylation and ciliary localization of SMO. This study highlights the mechanism and function of cholesterol modification of SMO.

中文翻译：

---

Smoothened 富含半胱氨酸结构域中的阳离子-π 相互作用对其胆固醇化和功能至关重要。

Hedgehog (Hh) 信号通路对于胚胎发育和组织更新至关重要。G 蛋白偶联受体 (GPCR) 样平滑蛋白 (SMO) 是 Hh 通路中的中央信号转导器。胆固醇与人 SMO 富含半胱氨酸结构域 (CRD) 的 D95 残基结合并共价连接。CRD 的胆固醇化对于 SMO 的激活至关重要。SMO 胆固醇化是一种 Ca 2+ 促进的自反应，需要在 D95 和 Y130 的侧链之间形成酯键作为中间体。目前尚不清楚 SMO 的其他残基是否参与 D95 与胆固醇之间的酯化反应。在本研究中，我们发现SMO-CRD(27-192)可以发生胆固醇化。除了 D95 和 Y130 之外，对胆固醇修饰至关重要的残基还包括 Y85、T88、T90、W109、W119、K133、E160 和 F166。T88、W109、W119 和 F166 似乎也参与蛋白质折叠。值得注意的是，我们发现 Y85 和 K133 形成阳离子-π 相互作用，其破坏消除了 SMO 的胆固醇化和纤毛定位。本研究重点阐述了SMO修饰胆固醇的机制和功能。