Immunodiversity of plant receptor kinase networks for synthetic circuit design

用于合成电路设计的植物受体激酶网络的免疫多样性

• 批准号: 10709286
• 负责人: Adam D Steinbrenner
• 金额: $ 37.93万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10709286
• 关键词: Agonist; Amino Acid Substitution; Animals; BAK1 gene; Binding; Binding Sites; Biotechnology; Cell Surface Receptors; DNA Library; Engineering; Epitopes; Evolution; G-Protein-Coupled Receptors; Gene Family; Genes; Genome; Genomics; Heterodimerization; Human; Human Engineering; Immune; Immune signaling; Immune system; Immunoglobulins; Immunologic Receptors; Inflammation; Leucine-Rich Repeat; Libraries; Life; Ligand Binding; Ligands; Medicine; Molecular Biology; Monitor; Natural Immunity; Orphan; Pathway interactions; Peptides; Phosphorylation; Phosphotransferases; Plant Model; Plants; Protein Kinase; Proteins; Receptor Activation; Receptor Gene; Reporter; Signal Pathway; Signal Transduction; Specificity; Structure; Toll-like receptors; Variant; Yeast Model System; design; extracellular; insight; network architecture; pathogen; protein aminoacid sequence; receptor; reconstruction; scaffold; sensor; synthetic construct; tool

PROJECT SUMMARY Immune systems across kingdoms of life recognize pathogen-associated molecules through germline-encoded innate immune receptors. Receptor repertoires in plants have evolved to detect an especially diverse set of ligands due to massive expansion of the receptor kinase gene family with specialized ligand recognition functions. Pairing receptor sequence diversity with specific recognition functions across 100 million RK genes (350,000 plant species * 500 receptors per genome) is a grand challenge in plant molecular biology. It also presents the opportunity to develop a new class of protein-based sensors for biotechnology. The Steinbrenner lab aims to characterize and deploy this vast plant immunodiversity for ligand-induced modulation of engineered signaling pathways. First, we will define the full ligand space that is monitored by plant receptors by focusing on the large subfamily of leucine-rich repeat receptor kinases (termed receptors here) which bind small peptide epitopes to initiate immune signaling. We will combine evolution- and structure-guided approaches to decode the basis of receptor:ligand specificity, including an extensive phylogenomic analysis, peptide variant libraries, and ancestral sequence reconstruction. We hypothesize that transitions in ligand specificity are marked by amino acid substitutions in predicted ligand binding sites among ancestral receptor genes. For “orphan” receptors lacking defined functions, we will conduct a genomic screen using synthetic DNA libraries encoding candidate pathogen epitopes using both plant and yeast models as reporters for receptor activation. We hypothesize that most receptors involved in plant innate immunity will be activated by specific pathogen-derived peptide sequences. Combined, these approaches will provide basic insights into receptor:ligand specificity as well as a toolkit of extracellular sensor domains responsive to specific peptide agonists. Second, we will leverage the unique network architecture of plant immune networks to engineer synthetic signaling pathways that do not interfere with endogenous animal signaling pathways. The plant receptors studied here signal through heterodimerization with a common co-receptor called BAK1. Co-receptor activation culminates in phosphorylation of substrates based on defined phosphocode motifs. We are currently engineering the human inflammation signaling pathway to accept orthogonal input from plant receptors by incorporating plant kinase substrates into specific, phosphoregulated signaling factors. In parallel, we will use plant receptor:co-receptor heterodimerization as a platform to scaffold endogenous human immune signaling domains from Toll-like receptors. We hypothesize that engineered pathways will allow modular tuning by diverse peptide ligands, providing an alternative to current immunoglobulin or GPCR-based synthetic tools. In summary our lab is poised to deploy tools for receptor de-orphanization and signaling pathway engineering to leverage the immense diversity driven by plant-pathogen co-evolution. (30 lines)

项目总结 不同生命王国的免疫系统通过生殖系编码识别病原体相关分子 先天免疫受体。植物中的受体谱系已经进化到检测到一组特别不同的 由于受体激酶基因家族的大规模扩展而产生的特殊配体识别 功能。在1亿个RK基因中配对具有特定识别功能的受体序列多样性 (350,000种植物*每个基因组500个受体)在植物分子生物学中是一个巨大的挑战。它还 介绍了为生物技术开发一类新的蛋白质传感器的机会。施泰因布伦纳 实验室的目标是表征和部署这种巨大的植物免疫多样性，用于配体诱导的调节 设计了信号通路。 首先，我们将通过关注大亚家族来定义由植物受体监控的完整的配基空间 富含亮氨酸的重复受体激酶(这里称为受体)，它结合小肽表位启动 免疫信号。我们将结合进化和结构指导的方法来解码 受体：配体特异性，包括广泛的系统基因组分析，多肽变异库，以及 祖先序列重建。我们假设配体专一性的转变是由氨基标记的。 祖先受体基因间预测的配基结合部位的酸取代。对于“孤儿”受体 由于缺乏明确的功能，我们将使用编码候选基因的合成DNA文库进行基因组筛选 使用植物和酵母模型作为受体激活报告的病原体表位。我们假设 大多数参与植物天然免疫的受体将被特定的病原体衍生的多肽激活 序列。结合起来，这些方法将提供对受体的基本见解：配体特异性以及 对特定多肽激动剂作出反应的细胞外感受器结构域工具包。 其次，我们将利用植物免疫网络的独特网络体系结构来设计合成 不干扰内源性动物信号通路的信号通路。植物受体 这里研究的信号是通过与一种共同的共受体BAK1发生异源二聚反应来实现的。共受体激活 根据定义的磷酸代码基序，底物的最终磷酸化。我们目前正在 设计人类炎症信号通路，通过以下方式接受植物受体的正交输入 将植物激酶底物加入到特定的、磷调节的信号因子中。同时，我们将使用 植物受体：共受体异源二聚化作为构建人体内源性免疫信号的平台 Toll样受体的结构域。我们假设，工程路径将允许模块化调整，通过 多样化的多肽配体，为目前的免疫球蛋白或基于GPCR的合成工具提供了一种替代。在……里面 摘要我们的实验室准备部署受体去孤儿和信号通路工程工具来 利用植物-病原体共同进化所驱动的巨大多样性。(30行)