Structural and Functional investigations of Hedgehog Signaling Transduction

Hedgehog 信号转导的结构和功能研究

• 批准号: 10473746
• 负责人: Xiaochun Li
• 金额: $ 30.78万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10473746
• 关键词: 25-hydroxycholesterol; Adult; Arbitration; Arrestins; Binding; Binding Proteins; Biochemical; Biochemistry; Biological; Biological Assay; C-terminal; CSNK1A1 gene; Cell Line; Cell Proliferation; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cellular biology; Cholesterol; Clinical; Clinical Trials; Complex; Cryoelectron Microscopy; Data; Development; Disease; Drug Prescriptions; Electron Microscopy; Embryo; Embryonic Development; Erinaceidae; Family; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; Genetic Transcription; Glioma; Goals; Grant; Homeostasis; Human; Hydroxycholesterols; Investigation; Kidney; Knock-out; Knowledge; Length; Ligands; Lipids; Malignant Neoplasms; Malignant neoplasm of pancreas; Mammals; Mass Spectrum Analysis; Mediating; Membrane Proteins; Modification; Molecular; Multiprotein Complexes; Mutagenesis; Oncogenes; Oncoproteins; Outcome; PTCH gene; Palmitates; Pathway interactions; Pertussis Toxin; Pharmacology; Pharmacotherapy; Phosphorylation; Phosphotransferases; Physiological; Play; Proteins; Regulation; Resolution; Role; SHH gene; Signal Pathway; Signal Transduction; Signaling Protein; Site; Skin Cancer; Structure; Tail; Tissues; Transcriptional Activation; X-Ray Crystallography; cellular transduction; cryogenics; hedgehog signal transduction; inhibitor; insight; malignant stomach neoplasm; metastatic colorectal; morphogens; new therapeutic target; novel; prevent; receptor; receptor function; recruit; smoothened signaling pathway; transcription factor; tumorigenesis

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.

项目概要 Hedgehog (HH) 蛋白是一个双脂质修饰形态发生素家族，可介导复杂的信号传导 HH信号通路在胚胎发育和成人中发挥着关键作用 组织稳态。以下发现进一步强调了 HH 途径的生理意义： 它的失调与多种人类癌症有关。 HH 信号被转导 通过与多胞体细胞表面受体 Patched1 (PTCH1) 结合； HH 增强了这种结合 共受体。 HH 与 PTCH1 的结合减轻了对卷曲类 G 蛋白偶联受体的抑制 平滑 (SMO)。激活的 SMO 随后会触发神经胶质瘤转录因子的激活 相关癌基因 (GLI)，可增强驱动细胞的 HH 途径靶基因的转录 增殖。为了阐明 HH 途径的分子基础，我们首先使用低温电子 显微镜（冷冻电镜）用天然棕榈酰化声波测定人 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 上的不同位点结合，产生 信号传导复合物。 HH 辅助受体如何促进 HH 与 PTCH 以及信号如何从 PTCH1 转导至 SMO，从而触发 HH 信号通路。 我们的初步研究表明，我们发现的氧甾醇 24(S), 25-环氧胆固醇 (24,25-EC) 与 PTCH1 结合，激活 SMO，从而允许招募启动 HH 的 G 蛋白 信号通路。基于我们的初步研究，我们现在准备：1）描绘机制 HH 辅助受体通过它增强 HH-PTCH1 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 相关蛋白在 HH 信号通路的调控。总的来说，这些研究将提供关于 HH 如何发生的重要见解。 在细胞膜上被识别并通过 SMO 响应性 G- 的作用调节基因转录 蛋白质。此外，我们的研究将具有重大的临床意义。 SMO 是 vismodegib 的靶标， 用于治疗皮肤癌的广泛处方药物，该药物也处于转移性临床试验阶段 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 信号通路中的靶标可用于治疗这些疾病。