Structure-based Bioengineering of Wnt Surrogates for Intestinal Stem Cell Biology and Therapy

摘要/摘要 WNT蛋白是一种在进化上保守的分泌型糖蛋白，调节细胞间的通讯 在发育和成体组织动态平衡过程中。WNT绑定到两个Frizzed，其中有结构 与G蛋白偶联受体和辅助受体LRP5/6相似，启动规范(即Wnt/b- 连结蛋白)信号，代表了研究最广泛的Wnt信号类型。我们和其他人展示了 肠上皮是一种典型的典型的Wnt/b-连环蛋白依赖组织，具有最终的共同 肠隐窝丢失的表型--关键是--Lgr5肠道干细胞(ISC)均匀丢失 通过抑制Wnt配体、它们的产生、受体或信号而明显。因为这一关键 在肠道和其他干细胞隔间的作用，Wnt信号轴是一个重要的治疗 靶向，但不良的生化特性阻碍了WNT作为再生药物的使用。我们 测定了Wnt与Frizzled型配体结合结构域(CRD)的第一个二元晶体结构。 这一突破提供了对WNT功能的不同见解。这揭示了Wnt/Fz的分子基础 交叉反应，并阐明了Wnt脂化位点及其在FZ结合中的关键作用(Science，2012)。 这个应用程序利用了Chris Garcia和Calvin Kuo在 Stanford(多PI)通过生物物理成像研究Wnt/受体相互作用的分子特征 方法，从而使人工生物工程WNT的基于结构的蛋白质工程成为可能 直接应用于肠道机制和治疗研究的类似物 上皮细胞和Lgr5ISC。目标1继续我们的结构性努力，以成像整个WNT/FrizzledLRP6三元组 跨膜复合体的X射线结晶学和冷冻电子显微镜研究 成功地表达和纯化了这种多分子组装。目标2专注于一个潜在的 我们取得了革命性的新发现，克服了将WNTS翻译成 治疗：1-天然WNTs因其脂化和2-FZ而难以表达为重组蛋白 交叉反应。我们已经开发出了水溶性的、FZ特定的替代Wnt激动剂，它可以模拟所有方面 但作为一种易于表达的非脂化重组蛋白。这些经过设计的代孕机器人WNT 激动剂不仅是生化上易于处理的基本Wnt信号获得和功能丧失的探针 机制，但为在再生医学中利用Wnt信号轴提供了一种新的策略，并将 在目标2中针对特定活性、FZ特异性和肠道器官生长进行了结构优化。最后，目标3 探索这些生物工程替代物支持Lgr5肠道干细胞的体内潜力， 减轻肠道辐射损伤和加强人体肠道器官移植。 总的来说，这项建议追求对Wnt结构和信号启动机制的基本分子见解， 并将这些信息应用于WNT研究和肠道生物学的实际问题。 好了！

ABSTRACT/SUMMARY Wnt proteins are phylogenetically conserved, secreted glycoproteins that regulate cell-cell communication during development and adult tissue homeostasis. Wnt binding to both Frizzled, which has structural similarities to G-protein coupled receptors, and to the co-receptors Lrp5/6, initiates canonical (i.e. Wnt/b- catenin) signaling, representing the most widely studied type of Wnt signaling. We and others demonstrated that the intestinal epithelium is an archetypal canonical Wnt/b-catenin-dependent tissue, with a final common phenotype of loss of intestinal crypts – and crucially – loss of Lgr5+ intestinal stem cells (ISC) uniformly apparent upon by inhibition of Wnt ligands, their production, receptors or signaling. Because of this critical role in the intestine and other stem cell compartments, the Wnt signaling axis is an important therapeutic target, but unfavorable biochemical properties have impeded the use of Wnt as a regenerative drug. We determined the first binary crystal structure of a Wnt in complex with a Frizzled ligand binding domain (CRD)— a breakthrough that offered diverse insights into Wnt function. This revealed the molecular basis for Wnt/Fz cross-reactivity, and elucidated the Wnt lipidation site and its essential role in Fz binding (Science, 2012). This application exploits a synergistic collaboration between Chris Garcia and Calvin Kuo at Stanford (Multi-PIs) to molecularly characterize Wnt/receptor interactions through biophysical imaging approaches, thus enabling the structure-based protein engineering of artificial bioengineered Wnt analogs which are directly applied to mechanistic and therapeutic investigation of the intestinal epithelium and Lgr5+ ISC. Aim 1 continues our structural efforts to image the entire Wnt/Frizzled/Lrp6 ternary transmembrane complex by X-ray crystallography and cryo-Electron Microscopy, building on preliminary successes in expressing and purifying this multimolecular assembly. Aim 2 focuses on a potentially transformative new discovery we have made that overcomes two major obstacles for translation of Wnts into therapeutics: 1- difficulty of expressing natural Wnts as recombinant proteins due to their lipidation and 2- Fz cross-reactivity. We have developed water-soluble, Fz-specific surrogate Wnt agonists that mimic all aspects of Wnt activity but as an easily expressed non-lipidated recombinant protein. These engineered surrogate Wnt agonists are not only biochemically tractable gain- and loss-of-function probes for basic Wnt signaling mechanisms, but offer a new strategy for exploiting the Wnt signaling axis in regenerative medicine and will be structurally optimized in Aim 2 for specific activity, Fz specificity and intestinal organoid growth. Lastly, Aim 3 explores the in vivo potential of these bioengineered surrogates for support of Lgr5+ intestinal stem cells, mitigation of intestinal radiation damage and augmented transplantation of human intestinal organoids. Collectively, this proposal pursues basic molecular insights into Wnt structure and signal initiation mechanisms, and applies this information to practical problems in Wnt research and intestinal biology. !