Structural and Functional investigations of Hedgehog Signaling Transduction

项目摘要 Hedgehog（HH）蛋白是一类介导复杂信号传导的双重脂质修饰的形态发生蛋白 HH信号通路，在胚胎发育和成年中起着关键作用。 组织内稳态HH通路的生理学意义进一步被以下发现所强调， 它的失调与多种类型的人类癌症有关。HH信号被转导 通过与多位细胞表面受体Patched 1（PTCH 1）结合; HH可增强这种结合 共受体HH与PTCH 1的结合缓解了卷曲类G蛋白偶联受体的抑制 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），其增强HH途径靶基因的转录，所述HH途径靶基因驱动细胞凋亡。 增殖为了阐明HH途径的分子基础，我们开始使用低温电子 显微镜（cryo-EM），以确定具有天然棕榈酰化声波的人PTCH 1蛋白的结构 刺猬（SHH）。这些研究揭示了HH的棕榈酸酯部分 促进其与PTCH 1的结合以及两个PTCH 1分子如何接合SHH上的不同位点， 信号能力复合体仍有待确定HH共受体如何促进HH与 PTCH以及信号如何从PTCH 1转导至SMO，从而触发HH信号通路。 我们的初步研究表明，氧化甾醇24（S），25-环氧胆固醇（24，25-EC），我们发现， 与PTCH 1结合，激活SMO，从而允许募集启动HH的G蛋白。 信号通路在我们初步研究的基础上，我们现在准备1）描绘机制 HH共受体通过其增强HH-PTCH 1结合; 2）在分子水平上探索24（S），25- EC调节SMO-Gi和SMO-Arrestin复合物; 3）阐明SMO-associated proteins在 HH信号通路的调节。总的来说，这些研究将提供关键的见解，HH是如何 在细胞膜上被识别，并通过SMO反应性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， 用于治疗皮肤癌的广泛处方药，其也处于转移性皮肤癌的临床试验阶段。 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.