Deciphering human signaling networks through synthetic activation of proteins in genomically recoded organisms with multiple open codons

通过具有多个开放密码子的基因组记录生物体中蛋白质的合成激活来破译人类信号网络

• 批准号: 10592390
• 负责人: Farren J. Isaacs
• 金额: $ 34.67万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-25 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592390
• 关键词: Acetylation; Amino Acids; Behavior; Binding Proteins; Biochemical; Biological Assay; Cell physiology; Cells; Chemicals; Code; Codon Nucleotides; Complex; Data; Development; Disease; Enzymes; Escherichia coli; Essential Genes; Evolution; Exhibits; Foundations; Genetic; Genetic Code; Genome engineering; Genomics; Goals; Human; Internet; Lysine; Mediating; Methods; Modality; Molecular; Names; Organism; Pattern; Phosphoproteins; Phosphorylation; Phosphoserine; Physiological; Positioning Attribute; Post-Translational Protein Processing; Protein Binding Domain; Protein Biosynthesis; Protein Dynamics; Protein Engineering; Proteins; Proteome; Proteomics; Recombinant Proteins; Recombinants; Regulation; Research; Ribosomes; Scaffolding Protein; Series; Serine; Set protein; Signal Transduction; Site; Specificity; Structure; System; Techniques; Technology; Terminator Codon; Testing; Translations; Work; fitness; genome editing; human disease; mutant; new technology; new therapeutic target; novel; novel strategies; novel therapeutics; protein activation; protein complex; protein protein interaction; reconstitution; release factor; synthetic construct; tool; yeast two hybrid system

Project Summary Healthy and diseased physiological states are governed by a complex web of interacting proteins that confer the collective behavior observed in cells. These protein networks are decorated with posttranslational modifications (PTMs) that determine their structure, function, and impart specificity for cellular signaling. Phosphorylation and acetylation represent two common PTMs that dictate healthy and disease states in human cells. For instance, 14-3-3 proteins scaffold thousands of important phosphoproteins with evidence suggesting that acetylation can modify its function. Current progress toward the elucidation of PTM-mediated signaling networks is hampered by the challenge of studying transient PTMs in cells and limited methods to produce proteins containing specific combinations of modified amino acids. Our previous efforts utilized a recoded E. coli strain (i.e., genomically recoded organism) to synthesize all human phosphoserine proteins using a genetic code expansion technique. We expanded this work through the development of a two hybrid like technology, named HI-P. HI-P validated previously observed phosphorylation dependent protein-protein interactions and identified scores of novel phosphoserine-mediated interactions across the human proteome that have been validated in biochemical- and cell-based assays. Our approach allows for synthetic DNA inputs to direct ribosome-based phosphoprotein synthesis and thus creates a programmable genetic tool to study the human phosphoproteome at the molecular level. Since deciphering complex protein networks require studying the impact of multiple PTMs in isolation and in combination, the key contribution of the proposed research is expected to expand the ability to genetically encode phosphoserine and acetylation at precise positions in 14-3-3 proteins to reveal PTM-mediated protein- protein interactions. Specific Aims: In this proposal, we seek to leverage a strong foundation of technologies, expertise, and preliminary data to construct a recoded E. coli with a single stop codon (two open codons) (Aim 1), develop a protein synthesis system capable of simultaneous encoding of phosphorylated and acetylated amino acids into proteins (Aim 2), and employ these capabilities to deconvolute PTM-mediated 14-3-3 protein network interactions (Aim 3). Significance: This work will be significant because it will enable the expression of programmable human proteins containing two PTMs thereby establishing a new approach to decipher complex human signaling networks at the molecular level. We anticipate this work will elucidate novel 14-3-3 protein network interactions governed by PTMs and enable new research into biomolecular and protein mechanisms that can be used to develop new therapies for human disease.

项目概要 健康和患病的生理状态由相互作用的蛋白质的复杂网络控制，这些蛋白质赋予 在细胞中观察到的集体行为。这些蛋白质网络经过翻译后修饰修饰 （PTM）决定其结构、功能并赋予细胞信号传导特异性。磷酸化和 乙酰化代表了决定人类细胞健康和疾病状态的两种常见 PTM。例如， 14-3-3 蛋白支撑着数千个重要的磷蛋白，有证据表明乙酰化可以 修改其功能。目前阐明 PTM 介导的信号网络的进展受到阻碍 通过研究细胞中瞬时 PTM 的挑战以及生产含有特定蛋白的有限方法 修饰氨基酸的组合。我们之前的努力利用了重新编码的大肠杆菌菌株（即基因组 重新编码的生物体）使用遗传密码扩展技术合成所有人类磷酸丝氨酸蛋白。 我们通过开发一种名为 HI-P 的两种混合技术来扩展这项工作。 HI-P 验证 先前观察到的磷酸化依赖性蛋白质-蛋白质相互作用并确定了新的分数 磷酸丝氨酸介导的跨人类蛋白质组的相互作用已在生物化学和生物化学中得到验证 基于细胞的测定。我们的方法允许合成 DNA 输入来引导基于核糖体的磷蛋白 合成，从而创建一个可编程遗传工具来研究人类磷酸化蛋白质组的分子水平 等级。由于破译复杂的蛋白质网络需要单独研究多个 PTM 的影响，并且 综上所述，拟议研究的关键贡献预计将扩大遗传研究的能力 在 14-3-3 蛋白的精确位置编码磷酸丝氨酸和乙酰化，以揭示 PTM 介导的蛋白- 蛋白质相互作用。具体目标：在本提案中，我们寻求利用强大的技术基础， 专业知识和初步数据来构建具有单个终止密码子（两个开放密码子）的重新编码的大肠杆菌（目标 1)、开发能够同时编码磷酸化和乙酰化的蛋白质合成系统 将氨基酸转化为蛋白质（目标 2），并利用这些功能对 PTM 介导的 14-3-3 蛋白质进行解卷积 网络交互（目标 3）。意义：这项工作意义重大，因为它将能够表达 含有两个 PTM 的可编程人类蛋白质，从而建立了一种破译复合物的新方法 分子水平上的人类信号网络。我们预计这项工作将阐明新型 14-3-3 蛋白质 由 PTM 控制的网络相互作用使生物分子和蛋白质机制的新研究成为可能 可用于开发人类疾病的新疗法。