Mechanisms of Smoothened Activation in Hedgehog Signaling

项目总结/摘要 刺猬信号的平滑激活机制。Hedgehog（Hh）信号 在人类和大多数后生动物中协调发育。在成体组织中，异常的Hh通路 激活导致各种癌症，包括皮肤基底细胞癌和小儿脑 髓母细胞瘤一种不寻常的机制使Hh信号转导。在基础状态下，传送器- 类似分子Patched 1（PTCH 1）抑制Smoothened（SMO），一种七跨膜蛋白。Hh抑制 PTCH 1，释放SMO活性，引起下游转录。一个主要的机械假说是 细胞固醇激活SMO，但在没有Hh的情况下被PTCH 1隔离。连接 PTCH 1对SMO激活的假定转运功能仍然是理解中的中心谜团之一 Hh信号是如何跨膜传递的一个关键的差距是如何，以及哪些，甾醇直接 影响SMO激活。本提案将从两个方面解决这些问题。第一个目标是 哪种固醇激活SMO。在这里，我们将结合联合收割机生物化学，结构生物学和信号研究， 定义哪些固醇可以激活SMO以及它们如何激活SMO。在第二个目标中，我们专注于甾醇如何结合 SMO上的不同位点影响Hh途径活性。我们会用X射线晶体学低温电子学 显微镜和核磁共振光谱法，以生成关于固醇在哪里的综合高分辨率视角 与SMO结合，以及它们如何影响SMO活性。这些研究将提供一个机械图片如何 Hh信号通过膜传导。这些见解可能使SMO抑制剂的设计更少 对肿瘤抗性敏感或用于再生医学的Hh通路调节剂。

PROJECT SUMMARY/ABSTRACT MECHANISMS OF SMOOTHENED ACTIVATION IN HEDGEHOG SIGNALING. The Hedgehog (Hh) signaling pathway coordinates development in humans and most metazoans. In adult tissues, aberrant Hh pathway activation drives various cancers, including basal cell carcinoma of the skin and pediatric brain medulloblastoma. An unusual mechanism enables Hh signal transduction. In the basal state, the transporter- like molecule Patched1 (PTCH1) represses Smoothened (SMO), a seven-transmembrane protein. Hh inhibits PTCH1, which unleashes SMO activity causing downstream transcription. A leading mechanistic hypothesis is that cellular sterols activate SMO, but are sequestered by PTCH1 in the absence of Hh. Connecting the putative transport function of PTCH1 to SMO activation remains one of the central mysteries in understanding how the Hh signal is transduced across the membrane. A critical gap is how, and which, sterols directly influence SMO activation. This proposal will address these questions in two aims. The first aim focuses on which sterols activate SMO. Here, we will combine biochemistry, structural biology, and signaling studies to define which sterols can activate SMO and how they do so. In the second aim, we focus on how sterol binding to distinct sites on SMO influences Hh pathway activity. We will use X-ray crystallography, cryo-electron microscopy, and NMR spectroscopy to generate an integrated high-resolution perspective on where sterols bind to SMO, and how they influence SMO activity. These studies will provide a mechanistic picture on how the Hh signal is transduced across the membrane. Such insights may enable the design of SMO inhibitors less susceptible to tumor resistance or Hh pathway modulators for regenerative medicine.