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)

项目摘要 跨越生命王国的免疫系统通过生殖系编码的基因识别病原体相关分子。 先天免疫受体植物中的受体库已经进化到检测一组特别多样化的 由于受体激酶基因家族的大规模扩展，配体具有专门的配体识别 功能协调发展的配对受体序列多样性与1亿个RK基因的特异性识别功能 (350 000个植物物种 * 每个基因组500个受体）是植物分子生物学的一个巨大挑战。它还 提供了开发用于生物技术的新型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 工程信号通路。 首先，我们将通过关注大的亚家族来定义由植物受体监测的完整配体空间 富含亮氨酸的重复受体激酶（此处称为受体）结合小肽表位， 免疫信号我们将结合联合收割机的进化和结构引导的方法来解码的基础， 受体：配体特异性，包括广泛的基因组分析，肽变体文库， 祖先序列重建我们假设配体特异性的转变是由氨基标记的， 在祖先受体基因中预测的配体结合位点中的酸取代。对于“孤儿”受体 由于缺乏明确的功能，我们将使用合成的DNA文库进行基因组筛选， 病原体表位，使用植物和酵母模型作为受体激活的报告基因。我们假设 大多数参与植物先天免疫的受体会被病原体衍生的特异性肽激活 序列的结合起来，这些方法将提供基本的见解受体：配体特异性以及 响应于特定肽激动剂的细胞外传感器结构域的工具包。 第二，我们将利用植物免疫网络的独特网络架构来设计合成 不干扰内源性动物信号传导途径的信号传导途径。植物受体 在这里研究的信号通过异源二聚体与共同的共同受体称为BAK 1。辅受体激活 在基于确定的磷酸基序的底物磷酸化中达到高潮。我们目前正在 工程化人类炎症信号传导途径以接受来自植物受体的正交输入， 将植物激酶底物整合到特定的磷酸调节信号因子中。同时，我们将使用 植物受体：共受体异源二聚化作为支架内源性人类免疫信号传导的平台 Toll样受体的结构域。我们假设，工程途径将允许模块调整， 不同的肽配体，为目前的免疫球蛋白或基于GPCR的合成工具提供了替代方案。在 摘要我们的实验室准备部署受体去磷酸化和信号通路工程的工具， 利用植物-病原体共同进化驱动的巨大多样性。(30线）