Sending and receiving Hedgehog and Wnt signals

项目摘要 Hedgehog（Hh）和Wnt信号通路是胚胎发生、成体干细胞分化和分化的关键信号通路。 维护，并深深参与癌症和出生缺陷。两种途径都是由脂质激活的- 修饰的分泌配体：Hh配体被棕榈酸酯和胆固醇修饰，而Wnt配体被 用棕榈酸酯改性。脂化对于Hh和Wnt配体的功能是必不可少的，但它使它们粘在一起。 这就提出了一个问题，即它们是如何从生产细胞中扩散的。 我们发现Hh通过与专用载体形成稳定的复合物而从细胞中释放 蛋白，Scube，这是一个由膜转运蛋白Dispatched催化的过程，它使用蛋白质的能量。 质膜钠梯度Scube然后将Hh传递到其受体，肿瘤抑制膜 蛋白质修补，触发特定的细胞反应。我们发现，令人惊讶的是，Scube阻止Hh的结合， 这就提出了Scube-Hh复合物如何激活信号传导的问题。我们发现， 细胞表面蛋白Cdon、Boc和Gas 1，已知为Hh的共受体，对于Scube的信号传导是必需的， Hh.具体地说，我们发现Hh共受体定义了一种新的顺序途径， Boc将Scube-Hh募集到细胞表面，之后Gas 1催化Hh从Scube转移到Patched。我们 还使用低温电子显微镜来确定Dispatched-Hh和Gas 1-Hh-Patched的结构 复合物，其分别在释放和递送过程中捕获Hh。最后，我们发现了两个 作为Wnt载体的分泌蛋白家族，解释了Wnt如何在可溶性 积极的形式。 我们的发现开启了关于Hh和Wnt信号传导的几个关键问题。我们建议结合 生物化学，结构，化学和细胞生物学，以实现以下目标： 1)确定Dispatched和Scube如何合作释放Hh配体 2)阐明Gas 1如何催化Hh-Patched复合物的形成，从而触发Hh信号传导 3)为了阐明Wnt配体是如何分泌的，然后递送到应答细胞， 这些研究很重要，原因如下：A）它们将促进对Hh的理解 和Wnt途径，通过阐明控制配体的关键途径; B）他们将鉴定新的靶点， 基于Hh和Wnt配体释放和递送的机制阻断癌症中的信号传导;以及C）我们的 新型化学探针将广泛应用于研究健康和疾病中的胆固醇和脂肪酸。

Project summary The Hedgehog (Hh) and Wnt signaling pathways are essential for embryogenesis, adult stem cell maintenance, and are deeply involved in cancer and birth defects. Both pathways are activated by lipid- modified secreted ligands: Hh ligands are modified with palmitate and cholesterol, while Wnt ligands are modified with palmitate. Lipidation is essential for the function of Hh and Wnt ligands, but it makes them stick strongly to membranes, raising the question of how they spread from producing cells. We discovered that Hh is released from cells by forming a stable complex with a dedicated carrier protein, Scube, a process catalyzed by the membrane transporter Dispatched, which uses the energy of the plasma membrane sodium gradient. Scube then delivers Hh to its receptor, the tumor suppressor membrane protein Patched, triggering specific cellular responses. We found that, surprisingly, Scube blocks binding of Hh to Patched, raising the question of how the Scube-Hh complex activates signaling. We discovered that three cell surface proteins Cdon, Boc and Gas1, known as co-receptors for Hh, are essential for signaling by Scube- Hh. Specifically, we showed that the Hh co-receptors define a novel sequential pathway whereby Cdon and Boc recruit Scube-Hh to the cell surface, after which Gas1 catalyzes Hh transfer from Scube to Patched. We also used cryo-electron microscopy to determine the structure of Dispatched-Hh and Gas1-Hh-Patched complexes, which capture Hh in the process of release and delivery, respectively. Finally, we discovered two families of secreted proteins that function as carriers for Wnts, explaining how Wnts are released in soluble and active form. Our findings open several critical questions about Hh and Wnt signaling. We propose combining biochemistry, structural, chemical and cell biology to accomplish the following aims: 1) To determine how Dispatched and Scube cooperate to release Hh ligands 2) To elucidate how Gas1 catalyzes formation of the Hh-Patched complex, to trigger Hh signaling 3) To elucidate how Wnt ligands are secreted and then delivered to responding cells These studies are important for the following reasons: A) They will advance understanding of the Hh and Wnt pathways, by elucidating critical pathways controlling the ligands; B) They will identify novel targets for blocking signaling in cancer, based on the mechanisms of Hh and Wnt ligand release and delivery; and C) Our novel chemical probes will be broadly applicable to study cholesterol and fatty acids in health and in disease.