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: 10447202
• 负责人: Kenan Christopher GARCIA
• 金额: $ 65.44万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447202
• 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; Transplantation; Variant; WNT Signaling Pathway; Water; Wnt proteins; X-Ray Crystallography; analog; antagonist; base; beta catenin; biophysical techniques; clinical translation; cross reactivity; dimer; frontier; gastrointestinal transplantation; imaging approach; improved; in vivo; in vivo evaluation; inhibitor; insight; intestinal crypt; intestinal epithelium; irradiation; loss of function; novel; novel therapeutics; palmitoylation; rational design; receptor; reconstitution; regenerative; scaffold; stem cell biology; stem cells; success; synergism; therapeutic evaluation; therapeutic target; therapeutically effective; tissue regeneration; translational barrier

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/总结 Wnt 蛋白是系统发育上保守的分泌性糖蛋白，可调节细胞间通讯 在发育和成体组织稳态过程中，Wnt 与 Frizzled 结合，具有结构性。 与 G 蛋白偶联受体和辅助受体 Lrp5/6 的相似性启动了规范（即 Wnt/b- 连环蛋白）信号，代表了我们和其他人证明的最广泛研究的 Wnt 信号类型。 肠上皮是典型的 Wnt/b-连环蛋白依赖性组织的原型，具有最终的共同点 肠隐窝丧失的表型 - 最重要的是 - Lgr5+ 肠干细胞 (ISC) 均匀丧失 由于这一关键，通过抑制 Wnt 配体、其产生、受体或信号传导而表现出来。 Wnt 信号轴在肠道和其他干细胞区室中发挥作用，是一种重要的治疗方法 目标，但不利的生化特性阻碍了 Wnt 作为再生药物的使用。 确定了 Wnt 与卷曲配体结合域 (CRD) 复合物的第一个二元晶体结构—— 这一突破为 Wnt 功能提供了多种见解，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 Chris Garcia 和 Calvin Kuo 之间的协同合作 斯坦福大学（Multi-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，从而实现人工生物工程 Wnt 的基于结构的蛋白质工程 直接应用于肠道机制和治疗研究的类似物 上皮细胞和 Lgr5+ ISC 目标 1 继续我们对整个 Wnt/Frizzled/Lrp6 三元体进行结构成像。 跨膜复合物通过 X 射线晶体学和冷冻电子显微镜，建立在初步 成功表达和纯化这种多分子组装体的目标是潜在的。 我们取得的革命性新发现克服了将 Wnts 转化为 治疗： 1- 由于其脂化和 2- Fz，难以将天然 Wnt 表达为重组蛋白 我们开发了可模拟所有方面的水溶性 Fz 特异性替代 Wnt 激动剂。 Wnt 活性，但作为一种易于表达的非脂化重组蛋白，这些工程化的替代 Wnt。 激动剂不仅是生化上易于处理的基本 Wnt 信号传导的功能获得和丧失探针 机制，但提供了在再生医学中利用 Wnt 信号轴的新策略，并将 在目标 2 中针对比活性、Fz 特异性和肠道类器官生长进行了结构优化。最后，在目标 3 中进行了结构优化。 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增强人体肠道类器官移植。 总的来说，该提案追求对 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。 ！

项目成果

Irreversible Electroporation for De-epithelialization of Murine Small Intestine.

用于小鼠小肠去上皮化的不可逆电穿孔。

• 发表时间: 2020-12
• 作者: Wood, Lauren S Y;Dunn, James C Y
• 通讯作者: Dunn, James C Y

Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling.

用于 Wnt 信号传导组织特异性增强的替代 R-spondins。

• 发表时间: 2020
• 影响因子: 3.7
• 作者: Luca, Vincent C;Miao, Yi;Li, Xingnan;Hollander, Michael J;Kuo, Calvin J;Garcia, K Christopher
• 通讯作者: Garcia, K Christopher

The Intestinal Stem Cell Niche: Homeostasis and Adaptations.

肠道干细胞生态位：稳态和适应。

• DOI: 10.1016/j.tcb.2018.08.001
• 发表时间: 2018-12
• 期刊: Trends in cell biology
• 影响因子: 19
• 作者: Santos AJM;Lo YH;Mah AT;Kuo CJ
• 通讯作者: Kuo CJ

Mutational signature in colorectal cancer caused by genotoxic pks+ E. coli.

由基因毒性 pks 大肠杆菌引起的结直肠癌的突变特征。

• 发表时间: 2020-04
• 影响因子: 64.8
• 作者: Pleguezuelos;Puschhof, Jens;Rosendahl Huber, Axel;van Hoeck, Arne;Wood, Henry M;Nomburg, Jason;Gurjao, Carino;Manders, Freek;Dalmasso, Guillaume;Stege, Paul B;Paganelli, Fernanda L;Geurts, Maarten H;Beumer, Joep;Mizutani, To
• 通讯作者: Mizutani, To

Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling.

间充质-上皮串扰通过 Wnt 和 Shh 信号传导塑造肠道区域化。

• 发表时间: 2022-02-07
• 影响因子: 16.6
• 作者: Maimets, Martti;Pedersen, Marianne Terndrup;Guiu, Jordi;Dreier, Jes;Thodberg, Malte;Antoku, Yasuko;Schweiger, Pawel J;Rib, Leonor;Bressan, Raul Bardini;Miao, Yi;Garcia, K Christopher;Sandelin, Albin;Serup, Palle;Jensen, Kim B
• 通讯作者: Jensen, Kim B