Structural and Functional investigations of Hedgehog Signaling Transduction

项目摘要 Hedgehog(HH)蛋白是一个双重脂质修饰的形态原家族，它介导一种复杂的信号转导 被称为HH信号通路，在胚胎发育和成年过程中起关键作用 组织动态平衡。研究结果进一步强调了HH途径的生理意义 它的失调与多种类型的人类癌症有关。HH信号被转换 通过它与多表位细胞表面受体Patched1(Ptch1)的结合，这种结合被HH增强 共同受体。HH与ptch1结合可解除Frizzled型G蛋白偶联受体的抑制 已平滑(SMO)。激活的SMO随后触发转录因子胶质瘤的激活- 相关癌基因(GLI)，促进驱动细胞的HH途径靶基因的转录 扩散。为了阐明HH途径的分子基础，我们从使用低温电子开始 用天然棕榈酰化声波显微镜(冷冻-EM)确定人ptch1蛋白的结构 刺猬(SHH)。这些研究揭示了HH的棕榈酸酯部分的分子基础 促进其与ptch1的结合，以及两个ptch1分子如何与SHH上的不同位置结合，产生 信令能力复合体。HH共受体如何促进HH与HH的结合仍有待确定 以及如何将信号从ptch1转导到SMO，从而触发HH信号通路。 我们的初步研究表明，我们发现的氧固醇24(S)，25-环氧胆固醇(24，25-EC) 与ptch1结合，激活SMO，从而允许启动HH的G蛋白的招募 信号通路。在我们初步研究的基础上，我们现在准备描述机制 2)在分子水平上探讨24(S)，25- EC调节SMO-GI和SMO-arrestin复合体；以及3)阐明SMO相关蛋白在 HH信号通路的调控。总而言之，这些研究将提供关于HH是如何的关键见解 在细胞膜上被识别，并通过SMO反应的G- 蛋白。此外，我们的研究将具有重要的临床意义。SMO是vismodegib的目标， 治疗皮肤癌的广泛处方药物，也处于转移的临床试验阶段 结直肠癌、晚期胃癌和胰腺癌。目前的研究可能会揭示新的治疗方法 HH信号通路中的靶点用于治疗这些疾病。

Project Summary Hedgehog (HH) proteins are a family of dual lipid-modified morphogens that mediate a complex signaling pathway known as the HH signaling pathway, which plays a key role in embryonic development and adult tissue homeostasis. The physiological significance of the HH pathway is further underscored by findings that its dysregulation has been implicated in multiple types of human cancers. The HH signal is transduced through its binding to the polytopic cell surface receptor Patched1 (PTCH1); this binding is enhanced by HH co-receptors. Binding of HH to PTCH1 relieves inhibition of the Frizzled-Class G-protein-coupled receptor Smoothened (SMO). Activated SMO subsequently triggers activation of the transcription factor glioma- associated oncogene (GLI), which enhances transcription of HH pathway target genes that drive cell proliferation. To elucidate molecular underpinnings of the HH pathway, we began by using cryogenic electron microscopy (cryo-EM) to determine the structure of human PTCH1 protein with native palmitoylated sonic hedgehog (SHH). These studies revealed the molecular basis through which the palmitate moiety of HH facilitates its binding to PTCH1 and how two PTCH1 molecules engage distinct sites on SHH, yielding a signaling-competent complex. It remains to be determined how HH co-receptors facilitate binding of HH to PTCH and how the signal is transduced from PTCH1 to SMO, triggering the HH signaling pathway. Our preliminary studies show that the oxysterol 24(S), 25-epoxycholesterol (24,25-EC), which we found associates with PTCH1, activates SMO and thereby permits recruitment of G proteins that initiate the HH signaling pathway. Building on our preliminary studies, we are now poised to 1) delineate mechanisms through which HH co-receptors augment HH–PTCH1 binding; 2) explore at the molecular level how 24(S),25- EC modulates SMO-Gi and SMO-Arrestin complexes; and 3) elucidate roles of SMO-associated proteins in regulation of the HH signaling pathway. Collectively, these studies will provide key insights into how HH is recognized at the cell membrane and modulates gene transcription through the action of a SMO-responsive G- protein. In addition, our studies will have significant clinical implications. SMO is the target of vismodegib, a widely prescribed drug for treatment of skin cancers that is also in the clinical trial stage for metastatic colorectal, advanced stomach, and pancreatic cancers. The current studies may reveal new therapeutic targets in the HH signaling pathway for treatment of these disorders.