Transducing Hedgehog signals across the plasma membrane

项目摘要 Hedgehog（Hh）信号通路对于胚胎发生、成体干细胞维持、 与出生缺陷和癌症有关在没有刺激的情况下，肿瘤抑制膜 蛋白Patched 1（Ptch 1）抑制7-羟色胺癌蛋白Smoothened（Smo），从而抑制Hh 信号该途径由Sonic Hedgehog配体（Shh）激活，该配体结合并抑制Ptch 1， 允许Smo变得活跃并触发下游信号事件。尽管这是至关重要的 Ptch 1和Smo的分子机制仍然不清楚：Ptch 1如何抑制Smo， Shh抑制Ptch 1，Smo如何被激活，以及它如何向下游传递信号。 我们最近发现胆固醇是长期寻找的内源性Smo激活剂，而Ptch 1 通过胆固醇控制Smo。我们解决了活性Smo的X射线结构，这提出了一种机制 胆固醇激活。这些结构也解释了一个典型的致癌Smo突变体的过度活跃 并指出Smo中可能涉及下游信令的部分。在初步工作中，我们发现 胆固醇从Smo快速转移到Ptch 1，提示Ptch 1抑制Smo的新机制。这 胆固醇转移被Shh阻断，通过Shh和Ptch 1之间的新型棕榈酸依赖性相互作用， 这是我们之前发现的，提示了Hh通路激活的简单机制。 我们建议利用生物化学、化学、细胞和结构生物学来实现以下目标： A）为了确定Ptch 1如何抑制Smo，以及Hh配体如何抑制Ptch 1，以触发Hh信号传导 B）为了精确地确定胆固醇如何激活Smo C）阐明Smo如何将Hh信号传递到细胞质 这些研究是重要的，原因如下：1）它们将促进我们对 Hh通路，通过破译关键信号传导机制; 2）他们将阐明Smo和Ptch 1突变 导致癌症; 3）他们将确定阻断致癌Hh信号传导的新靶点，基于 Smo和Ptch 1的作用机制; 4）我们开发的用于研究Shh和Ptch 1的新型脂质探针 将广泛适用于Hh途径之外。

Project summary The Hedgehog (Hh) signaling pathway is essential for embryogenesis, for adult stem cell maintenance, and is involved in birth defects and cancer. In the absence of stimulation, the tumor suppressor membrane protein, Patched1 (Ptch1), inhibits the seven-spanner oncoprotein Smoothened (Smo), thus inhibiting Hh signaling. The pathway is activated by the Sonic Hedgehog ligand (Shh), which binds and inhibits Ptch1, allowing Smo to become active and to trigger downstream signaling events. In spite of the critical importance of Ptch1 and Smo, their molecular mechanisms remain obscure: it is unknown how Ptch1 inhibits Smo, how the Shh inhibits Ptch1, how Smo is activated, and how it relays signals downstream. We recently discovered that cholesterol is the long-sought endogenous Smo activator, and that Ptch1 controls Smo via cholesterol. We solved X-ray structures of active Smo, which suggested a mechanism for activation by cholesterol. The structures also explained the hyperactivity of a classical oncogenic Smo mutant and pointed to a portion in Smo likely involved in downstream signaling. In preliminary work, we discovered rapid cholesterol transfer from Smo to Ptch1, suggesting a novel mechanism for Smo inhibition by Ptch1. This cholesterol transfer is blocked by Shh, via the novel palmitate-dependent interaction between Shh and Ptch1, which we previously discovered, suggesting a simple mechanism for Hh pathway activation. We propose to use biochemistry, chemical, cell and structural biology to accomplish the following aims: A) To determine how the Ptch1 inhibits Smo, and how the Hh ligand inhibits Ptch1, to trigger Hh signaling B) To determine precisely how cholesterol activates Smo C) To elucidate how Smo relays Hh signals to the cytoplasm These studies are important for the following reasons: 1) They will advance our understanding of the Hh pathway, by deciphering critical signaling mechanisms; 2) They will clarify how Smo and Ptch1 mutations cause cancer; 3) They will identify novel targets for blocking oncogenic Hh signaling, based on the mechanisms of Smo and Ptch1; and 4) The novel lipid probes that we developed for studying Shh and Ptch1 will be broadly applicable beyond the Hh pathway.