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

用于肠干细胞生物学和治疗的 Wnt 替代物的基于结构的生物工程

• 批准号: 10197113
• 负责人: Kenan Christopher GARCIA
• 金额: $ 66.95万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10197113
• 关键词: Address; Adult; Affinity; Agonist; Binding; Biochemical; Biological Availability; Biological Process; Biology; Biomedical Engineering; Biophysics; Cell Communication; Cell Compartmentation; Cell Surface Receptors; Cell Therapy; Cells; Collaborations; Complex; Cryoelectron Microscopy; Crystallization; Cysteine-Rich Domain; Dependence; Development; Dimerization; Drug Kinetics; Electron Microscopy; Embryonic Development; Engineering; G-Protein-Coupled Receptors; Generations; Geometry; Glycoproteins; Goals; Growth; Growth Factor; Heterodimerization; Histologic; Homeostasis; Human; Hydrophobicity; Image; In Vitro; Intestines; Investigation; LGR5 gene; Length; Ligand Binding Domain; Ligands; Lipids; Mediating; Molecular; Mus; Natural regeneration; Nature; Organoids; Pharmaceutical Preparations; Phenotype; Phylogenetic Analysis; Production; Property; Protein Engineering; Radiation induced damage; Receptor Signaling; Recombinant Proteins; Regenerative Medicine; Research; Research Design; Role; Science; Signal Pathway; Signal Transduction; Site; Specificity; Structure; Testing; Therapeutic; Tissues; Translating; Translations; Transplantation; Variant; WNT Signaling Pathway; Water; Wnt proteins; X-Ray Crystallography; analog; base; beta catenin; biophysical techniques; clinical translation; cross reactivity; design; dimer; frontier; gastrointestinal transplantation; imaging approach; improved; in vivo; in vivo evaluation; inhibitor/antagonist; insight; intestinal crypt; intestinal epithelium; irradiation; loss of function; novel; novel therapeutics; palmitoylation; receptor; reconstitution; regenerative; scaffold; stem cell biology; stem cells; success; synergism; therapeutic evaluation; therapeutic target; therapeutically effective; tissue regeneration

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. !

摘要/摘要 Wnt蛋白质是系统发育保守的，分泌的糖蛋白，调节细胞 - 细胞通信 在发育和成人组织稳态期间。 wnt与两者都有结构的毛躁结合 与G蛋白偶联受体的相似性，与受体LRP5/6相似，启动了规范（即Wnt/B- Catenin）信号，代表WNT信号的最广泛研究类型。我们和其他人证明了 肠上皮是一种原型的典型Wnt/b-catenin依赖性组织，最终常见 肠道隐窝丧失的表型，并且完全 - 统一的LGR5+肠干细胞（ISC）丧失 显然，抑制Wnt配体，其产生，接收器或信号传导。因为这个关键 在肠道和其他干细胞室中的作用，Wnt信号轴是重要的治疗方法 靶标，但不利的生化特性阻碍了Wnt用作再生药物的使用。我们 确定了Wnt在复合物中的第一个二元晶体结构，该结构具有卷曲的配体结合结构域（CRD） - 一个突破，为潜水员提供了对Wnt功能的见解。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）。 该应用程序利用了克里斯·加西亚（Chris Garcia）和加尔文·库（Calvin Kuo）之间的协同合作 斯坦福（多核）以分子表征Wnt/受体相互作用通过生物物理成像 方法，从而实现了人工生物工程Wnt的基于结构的蛋白质工程 直接应用于肠道机械和治疗研究的类似物 上皮和LGR5+ ISC。 AIM 1继续我们的结构性努力来对整个Wnt/毛躁/LRP6三元 X射线晶体学和冷冻电子显微镜的跨膜复合物，建立在初步的基础上 在表达和纯化该多分子组件方面取得了成功。 AIM 2专注于潜在的 我们做出的变革性新发现克服了将Wnts翻译成的两个主要障碍 治疗：1-由于其脂质和2- fz而表达天然WNT作为重组蛋白的困难 交叉反应性。我们已经开发了水溶性，Fz特异性的替代wnt wnt激动剂，它们模仿了所有方面 Wnt活性的活性，但作为一种易于表达的非脂化重组蛋白。这些设计的代理Wnt 激动剂不仅是生物化学上可触及的基本Wnt信号的功能丧失问题 机制，但提供了一种新的策略来利用再生医学中的Wnt信号轴，将是 在AIM 2中对特定活性，FZ特异性和肠癌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的体内潜力，以支持LGR5+肠干细胞， 缓解肠道辐射损伤和人类肠癌的移植增强。 总的来说，该建议追求基本的分子见解，对Wnt结构和信号启动机制， 并将这些信息应用于WNT研究和肠生物学的实际问题。 呢