Structural Basis of Norrin-induced Wnt/beta-catenin signaling

 描述(申请人提供)：Norrin，也被称为Norrie病蛋白或X连锁渗出性玻璃体视网膜病变2蛋白，是一种分泌型视网膜生长因子，具有血管生成和神经保护特性。Norrin基因突变与Norrie病、常见的渗出性玻璃体视网膜病变(FEVR)、早产儿视网膜病变(ROP)和其他视网膜血管减少疾病有关。尽管它的结构与Wnt蛋白不同，但Norrin与Wnt受体Frizzled4(Fz4)和Wnt共受体LRP5/6特异结合，并激活细胞内典型的Wnt/-catenin信号通路。Norrin如何通过与Fz4和LRP5/6的相互作用激活Wnt/-Catenin信号通路，以及Norrin如何受到其他蛋白质的调控仍然是一个谜。在这里，我们旨在通过结合X射线结晶学、冷冻-EM和生化分析测定去甲肾上腺素与Fz4和/或LRP5/6的三维结构，来揭示去甲肾上腺素依赖的Wnt途径激活的结构基础。我们还将提供关键线索，以了解去甲肾上腺素是如何受到Wnt途径调节因子，如LGR4的调节。了解Norrin诱导的Wnt通路激活的结构基础不仅将揭示Norrin信号机制，而且有助于开发预防和/或治疗视网膜血管减少疾病的新方法，仅在美国就有大约2000万患者受到影响。此外，由于Wnt蛋白因其必需的脂类修饰而溶解性差，阻碍了其在治疗中的实际应用，未经任何翻译后修饰的重组Norrin蛋白在激活规范的Wnt信号方面完全活跃。因此，我们的工作也可能为设计可用于干细胞生物学和再生医学的可溶性Wnt模拟物提供一个结构模板。

 DESCRIPTION (provided by applicant): Norrin, also known as the Norrie disease protein or X-linked exudative vitreoretinopathy 2 protein, is a secreted retinal growth factor with angiogenic and neuroprotective properties. Norrin mutations are associated with the Norrie disease, familiar exudative vitreoretinopathy (FEVR), retinopathy of prematurity (ROP), and other retinal hypovascularization diseases. Despite its distinct structure from Wnt proteins, Norrin binds specifically to Frizzled 4 (Fz4), a Wnt receptor, and Lrp5/6, a Wnt co-receptor, and activates the canonical Wnt/-catenin signaling pathway inside the cell. How Norrin activates the Wnt/-catenin signaling pathway through its interactions with Fz4 and Lrp5/6, and how Norrin is regulated by other proteins, remain enigmatic. Here we aim to unravel the structural basis of Norrin-dependent Wnt pathway activation through the determination of the 3D structures of Norrin in complex with Fz4 and/or Lrp5/6, using a combination of X-ray crystallography, cryo-EM and biochemical analysis. We will also provide key clues to understand how Norrin is regulated by Wnt pathway regulators such as Lgr4. Understanding the structural basis of Norrin-induced Wnt pathway activation will not only reveal Norrin signaling mechanism, but also help with the development of new approaches for prevention and/or treatment of retinal hypovascularization diseases that affects about 20 million patients in the United States alone. In addition, in contras to the poor solubility of Wnt proteins due to their essential lipid modification, which prevented their practical uses in therapeutics, recombinant Norrin protein without any posttranslational modification is fully active in activating canonical Wnt signaling. Therefore, our work may also provide a structural template for designing soluble Wnt mimetics useful for stem cell biology and regenerative medicine.