The structure-based traceless specific fluorescence labeling of the smoothened receptor
The structure-based traceless specific fluorescence labeling of the smoothened receptor
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基于结构的平滑受体的无痕特异性荧光标记
DOI：
10.1039/c9ob00654k
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发表时间：
2019
影响因子：
3.2
通讯作者：
Tao Houchao
中科院分区：
文献类型：
--
作者：
Xue Dongxiang;Ye Lintao;Zheng Jun;Wu Yiran;Zhang Xianjun;Xu Yueming;Li Tao;Stevens Raymond C;Xu Fei;Zhuang Min;Zhao Suwen;Zhao Fei;Tao Houchao
平滑受体（SMO）介导刺猬蛋白（Hh）信号通路，在胚胎发育和肿瘤发生中发挥重要作用。 SMO 的可视化有可能为其神秘机制和相关疾病发病机制提供新的见解。基于SMO结构研究的最新进展，我们设计并表征了一组亲和探针，以促进SMO在K395的ε-胺上的开启荧光标记。这些化学探针是通过与小的非荧光单元 O-硝基苯并恶二唑 (O-NBD) 缀合而衍生自有效的 SMO 拮抗剂骨架。在这种情况下，我们开发了最佳探针，能够有效、选择性地点亮 SMO，无论它是在胶束中还是在天然膜中。更重要的是，所得的标记 SMO 仅带有非常小的荧光团，并允许通过残余配体模块的解离来恢复未占据的口袋。这些优点应该使探针能够成为监测 SMO 运输、了解 Hh 激活机制、甚至未来诊断肿瘤发生的潜在工具。
The smoothened receptor (SMO) mediates the hedgehog (Hh) signaling pathway and plays a vital role in embryonic development and tumorigenesis. The visualization of SMO has the potential to provide new insights into its enigmatic mechanisms and associated disease pathogenesis. Based on recent progress in structural studies of SMO, we have designed and characterized a group of affinity probes to facilitate the turn-on fluorescence labeling of SMO at the ε-amine of K395. These chemical probes were derived from a potent SMO antagonist skeleton by the conjugation of a small non-fluorescent unit, O-nitrobenzoxadiazole (O-NBD). In this context, optimal probes were developed to be capable of efficiently and selectively lighting up SMO regardless of whether it is in micelles or in native membranes. More importantly, the resulting labeled SMO only bears a very small fluorophore and allows for the recovery of the unoccupied pocket by dissociation of the residual ligand module. These advantages should allow the probe to serve as a potential tool for monitoring SMO trafficking, understanding Hh activation mechanisms, and even the diagnosis of tumorigenesis in the future.