Molecular Principles of Wnt-Frizzled Signal Initiation and Inhibition

描述(申请人提供)：WNT蛋白是在系统发育上保守的、分泌的糖蛋白，通过规范和非规范的信号通路调节胚胎发育期间的细胞间通讯和成人组织的动态平衡。由于WNT信号在癌症的发生和发展、组织更新和干细胞分化中的中心作用和治疗意义，WNT信号被广泛研究。Wnt与Frizzed的结合在结构上与G蛋白偶联受体和辅助受体LRP5/6有相似之处，对于Wnt/-catenin信号转导是必不可少的，并被多种内源性拮抗剂所抵消。在这项建议中，我们解决了完全缺乏关于Wnts的细胞外结构信息，Wnts与FrizzledR复合的方式，以及Wnt和FrizzledWIF，DKK和Kreman等内源抑制蛋白的相互作用。鉴于Wnt信号在多种人类疾病中的核心重要性，结构信息现在变得至关重要，以便清楚地描述受体-配体特异性的基础和受体激活的机制。目前该领域的一个困惑是，Wnts、FZ和LRP6是高度交叉反应的，使特定的生物学表型与特定的Wnt-Fz受体对的归属复杂化。Wnt-受体复合体的结构可以通过可视化简并与配体特定的接触以及更高阶信号复合体的结构来帮助解决这个问题。然而，获取结构数据的一个主要技术障碍是Wnt蛋白含有疏水的脂类修饰，这使得Wnt的过度表达和结晶变得复杂。在这个建议中，我们利用各种新的(酵母展示)和传统的(X射线结晶学)方法来获得参与受体激活和抑制的细胞外Wnt复合体的三维结构。我们的目标是重组几个重组的Wnt-FrizzledLRP5/6和Wnt-拮抗剂络合物，以表征它们的组装、亲和力和化学计量。我们还将尝试一项高度创新的实验，通过体外进化来生产水溶性、非棕榈酰化、生物活性的WNTs。利用X射线结晶学，我们希望阐明WNT-FrizzledLRP5/6识别的结构原理，以及拮抗剂WIF、DKK和Kreman抑制Wnt-FrizzledLRP5/6复合体的形成，并与WNT领域的知名合作者一起，将结构数据转化为功能洞察力的实验。最终，这些雄心勃勃的研究结果将对揭示新的蛋白质工程策略以讯问和治疗靶向Wnt信号具有价值。 与公共健康相关：WNTS是一种分泌型生长因子，通过与FrizzledG蛋白偶联受体以及各种辅助受体和拮抗剂相互作用，在胚胎发育和成人组织再生中发挥作用。WNT信号代表了癌症治疗干预、组织更新和干细胞分化的重要新前沿。我们希望获得有关Wnt-受体复合体的组装和三维结构的信息，以了解Wnt信号转导机制，并揭示新的蛋白质工程策略，用于临床靶向Wnt相关疾病。

DESCRIPTION (provided by applicant): Wnt proteins are phylogenetically conserved, secreted glycoproteins that regulate cell-to-cell communication during embryonic development and adult tissue homeostasis through canonical and non-canonical signaling pathways. Wnt signaling is intensively studied due to its central role, and therapeutic implications, in the development and progression of cancer, tissue renewal, and differentiation of stem cells. Wnt binding to Frizzled, which has structural similarities to G-protein coupled receptors, and the co-receptors Lrp5/6, is indispensable for Wnt/-catenin signaling, and is counterbalanced by a variety of endogenous antagonists. In this proposal we address the complete lack of extracellular structural information on Wnts, the manner in which Wnts complex with Frizzled receptors, as well as Wnt and Frizzled interactions with endogenous inhibitory proteins such as WIF, Dkk and Kremen. Given the central importance of Wnt signaling for a variety of human diseases, structural information has now become critical in order to clearly delineate the basis of receptor- ligand specificity and mechanisms of receptor activation. A current confusion in this field is that Wnts, Fz and Lrp6 are highly cross-reactive, complicating the attribution of specific biological phenotypes to specific Wnt-Fz receptor pairs. Structures of Wnt-receptor complexes could help solve this problem by visualizing degenerate versus ligand-specific contacts, as well as the architectures of the higher order signaling complexes. However, a major technical obstacle to obtaining structural data has been that Wnt proteins contain a hydrophobic lipid modification that complicates Wnt over-expression and crystallization. In this proposal we utilize a variety of novel (yeast display) and traditional (X-ray crystallography) methodologies to obtain three-dimensional structures of extracellular Wnt complexes involved in receptor activation, and inhibition. We aim to reconstitute several recombinant Wnt-Frizzled, Wnt-Frizzled-Lrp5/6 and Wnt-antagonist complexes, in order to characterize their assemblies, affinities and stoichiometries. We will also attempt a highly innovative experiment to produce water-soluble, non-palmitoylated, bioactive Wnts by in vitro evolution. Using X-ray crystallography, we hope to elucidate the structural principles of Wnt-Frizzled-Lrp5/6 recognition, and inhibition of Wnt-Frizzled-Lrp5/6 complex formation by the antagonists WIF, DKK and Kremen, and together with established collaborators in the Wnt field, translate the structural data into functionally insightful experiments. Ultimately the results of these ambitious studies will be valuable for revealing new protein engineering strategies to interrogate and therapeutically target Wnt signaling. PUBLIC HEALTH RELEVANCE: Wnts are secreted growth factors that exert their actions in embryonic development and adult tissue regeneration through interactions with Frizzled G-protein coupled receptors, in concert with a variety of co- receptors and antagonists. Wnt signaling represents an important new frontier for therapeutic intervention in cancer, tissue renewal, and differentiation of stem cells. We wish to acquire information about the assembly and three-dimensional structures of Wnt-receptor complexes in order to understand Wnt signaling mechanisms, and reveal new protein engineering strategies to clinically target Wnt-associated diseases.