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

摘要/总结 Wnt蛋白是一种高度保守的分泌型糖蛋白，调节细胞间的通讯 在发育和成年组织内环境稳定期间。Wnt与卷曲蛋白结合，卷曲蛋白具有结构性 与G-蛋白偶联受体和共受体Lrp 5/6的相似性，启动典型的（即Wnt/B- 连环蛋白）信号传导，代表了最广泛研究的Wnt信号传导类型。我们和其他人证明了 肠上皮是典型的典型Wnt/β-连环蛋白依赖性组织， 肠隐窝丢失的表型-并且至关重要的是-Lgr 5+肠干细胞（ISC）均匀丢失 通过抑制Wnt配体、其产生、受体或信号传导而明显。由于这一关键 在肠道和其他干细胞区室中的作用，Wnt信号传导轴是重要的治疗性细胞因子。 目标，但不利的生物化学特性阻碍了Wnt作为再生药物的使用。我们 确定了Wnt与卷曲配体结合结构域（CRD）复合的第一个二元晶体结构， 这一突破为Wnt功能提供了不同的见解。这揭示了Wnt/Fz的分子基础 交叉反应性，并阐明了Wnt脂化位点及其在Fz结合中的重要作用（Science，2012）。 这个应用程序利用了Chris Garcia和卡尔文郭之间的协同合作， 斯坦福大学（多PI）通过生物物理成像对Wnt/受体相互作用进行分子表征 方法，从而使基于结构的蛋白质工程的人工生物工程Wnt 直接应用于肠道疾病的机制和治疗研究的类似物 上皮和Lgr 5 + ISC。目标1继续我们的结构努力，以成像整个Wnt/Frizzled/Lrp 6三元 通过X射线晶体学和冷冻电子显微镜，建立在初步的跨膜复合物 成功表达和纯化这种多分子组装体。目标2关注潜在的 我们已经取得了一个变革性的新发现，克服了将Wnt翻译成 治疗：1-由于其脂化和2- Fz，天然Wnt难以表达为重组蛋白 交叉反应性我们已经开发了水溶性的Fz特异性替代Wnt激动剂， 但作为一种容易表达的非脂化重组蛋白。这些工程替代Wnt 激动剂不仅是基本Wnt信号传导的生物化学易处理的功能获得和丧失探针 机制，但提供了一个新的战略，利用Wnt信号轴在再生医学， 在目标2中针对比活性、Fz特异性和肠类器官生长进行了结构优化。最后，目标3 探索了这些生物工程替代物支持Lgr 5+肠干细胞的体内潜力， 减轻肠辐射损伤和增强人肠类器官的移植。 总的来说，这项提案追求对Wnt结构和信号起始机制的基本分子见解， 并将这些信息应用于Wnt研究和肠道生物学中的实际问题。 !

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. !