Mechanisms of Smoothened Activation in Hedgehog Signaling

项目摘要/摘要 刺猬信号的平滑激活机制。刺猬(HH)信号 途径协调人类和大多数后生动物的发育。在成人组织中，异常的HH途径 激活会导致多种癌症，包括皮肤基底细胞癌和儿童脑癌 髓母细胞瘤。一种不寻常的机制使HH信号转导成为可能。在基本状态下，转运体- 与Patched1(Ptch1)分子一样，ptch1抑制七跨膜蛋白Smoothens(SMO)。HH抑制 Ptch1，释放SMO活性，导致下游转录。一个主要的机械论假说是 细胞甾醇激活SMO，但在没有HH的情况下被ptch1隔离。连接 Ptch1到SMO激活的运输功能仍然是理解的中心谜团之一。 HH信号是如何通过细胞膜传递的。一个关键的差距是如何以及哪种甾醇直接 影响SMO的激活。这项提案将从两个方面解决这些问题。第一个目标集中在 哪种甾醇能激活SMO。在这里，我们将结合生物化学、结构生物学和信号研究来 确定哪些甾醇可以激活SMO，以及它们是如何激活SMO的。在第二个目标中，我们将重点放在如何结合甾醇 SMO上不同的位置会影响HH途径的活性。我们将使用X射线结晶学，低温电子 显微镜和核磁共振光谱学，以生成关于甾醇在哪里的综合高分辨率透视 绑定到SMO，以及它们如何影响SMO活动。这些研究将提供一幅机械图景，说明 HH信号通过细胞膜进行传递。这种见解可能会使SMO抑制剂的设计变得更少 对肿瘤耐药或再生医学中的HH途径调节剂敏感。

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.