Molecular mechanisms of Hedgehog receptor function

项目摘要 Hedgehog(HH)信号指定许多后生动物器官的胚胎组织模式并维持这种模式 通过调控增殖或分化诱导信号的表达实现胚胎后的组织模式 以成体组织干细胞或祖细胞为靶细胞。基于我们的研究，开发了用于阻断HH途径活性的药物 之前的工作，已经获得FDA批准用于治疗外胚层来源的癌症，如基底细胞 癌症。在胰腺癌、膀胱癌和其他起源于内胚层的癌症中，肿瘤中的HH途径活性- 相关间质是肿瘤生长和进展的障碍，因此提示途径激活。 而不是抑制作为一种治疗方法。此外，通路的激活在 骨骼和肌肉修复，减少与炎症性肠病相关的病理，并防止 或减轻损伤和血脑屏障的破坏，以及其他新兴的生物活动。论 另一方面，慢性低水平的肺通路活性升高，与AS的表达减少有关 HH途径抑制因子HHIP(HH相互作用蛋白)与慢性阻塞性肺疾病有遗传关联 慢性阻塞性肺病(COPD)是全球第三大死亡原因。 在这个项目的前一个资助期，我们使用了蛋白质结构测定和生化 以及细胞生物学方法来建立HH信号的分子机制，其中HH与其 受体Patched1(Ptch1)通过减轻ptch1介导的对必要的 换能器和GPCR族成员，光滑(Smo)。我们发现胆固醇是 Ptch1和Smo，膜内叶中的胆固醇通过ptch1转运活性而降低， 并且与PTCH1的HH绑定阻止该传输活动。这些事件关键地调节通路的活动，因为 Smo的构象转换到其活性状态需要从Smo的内叶进入并结合 在光滑的七跨膜束内形成一个中央空腔。我们还展示了如何 Dispatched1(Disp1)运输机在结构上与PTCH1相关，使用钠熔剂为其出口和包装提供动力 双脂修饰的Sonic Hedgehog蛋白信号(ShhNp)，使其能够作为一种 可溶性形态原与其载体Scube2形成络合物。我们建议在这里加深我们对HH的理解 通过建立能量来源和分步脂质来进行信号转导和通路调节。 Ptch1和Disp1转运体的处理机制。我们将确定ShhNp：Scube2的结构 并对其从Disp1释放的机制进行了研究。最后，我们计划阐明HH的机制。 HHIP的信号拮抗作用，用低温电子显微镜确定高分辨结构并对其进行功能解剖 膜相关帐篷状HHIP多聚体，封闭HH的所有受体相互作用表面 蛋白。我们的发现可能为治疗调节HH途径活性的新方法提供基础。

Project Summary Hedgehog (Hh) signaling specifies the embryonic tissue pattern of many metazoan organs and maintains this tissue pattern post-embryonically by regulating the expression of proliferation- or differentiation-inducing signals that target adult tissue stem or progenitor cells. Drugs developed to block Hh pathway activity, based on our previous work, have received FDA approval for treatment of ectodermally-derived cancers, such as basal cell carcinoma. In pancreatic, bladder, and other cancers of endodermal origin, Hh pathway activity in tumor- associated stroma presents a barrier to tumor growth and progression, thus suggesting pathway activation rather than inhibition as a therapeutic approach. In addition, pathway activation has a beneficial regenerative role in bone and muscle repair, in reducing pathology associated with inflammatory bowel disease, and in preventing or ameliorating injury and breach of the blood-brain-barrier, among other emerging biological activities. On the other hand, chronic low-level elevation of pathway activity in the lung, as is associated with reduced expression of the Hh pathway inhibitor Hhip (Hh-interacting protein), is genetically linked to chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide. During the previous funding period for this project we utilized protein structure determination and biochemical and cell biological approaches to establish the molecular mechanism of Hh signaling, in which Hh binding to its receptor Patched1 (Ptch1) activates the pathway by alleviating Ptch1-mediated suppression of the essential transducer and GPCR family member, Smoothened (Smo). We found that cholesterol is the crucial link between Ptch1 and Smo, that cholesterol in the inner leaflet of the membrane is decreased by Ptch1 transport activity, and that Hh binding to Ptch1 blocks this transport activity. These events critically regulate pathway activity, as conformational switching of Smo to its active state requires entry and binding of a sterol from the inner leaflet of the membrane into a central cavity within the Smoothened seven-transmembrane bundle. We also showed how the Dispatched1 (Disp1) transporter, structurally related to Ptch1, uses Na+ flux to power its export and packaging of the dually lipid modified Sonic hedgehog protein signal (ShhNp), enabling it to move through tissues as a soluble morphogen in complex with its carrier Scube2. We propose here to deepen our understanding of Hh signal transduction and pathway regulation by establishing the energy sources and the step-by-step lipid- handling mechanisms of the Ptch1 and Disp1 transporters. We will determine the structure of the ShhNp:Scube2 morphogen, and the mechanism of its release from Disp1. Finally we plan to elucidate the mechanism of Hh signal antagonism by Hhip, using cryo-EM to determine the high-resolution structure and functionally dissect a membrane-associated tent-like Hhip multimeric complex that occludes all receptor-interacting surfaces of the Hh protein. Our findings may provide a basis for new approaches to therapeutic modulation of Hh pathway activity.