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: 10207998
• 负责人: Farren J. Isaacs
• 金额: $ 35.82万
• 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/10207998
• 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; Mediating; Methods; Modality; Molecular; Names; Organism; Pattern; Phosphoproteins; Phosphorylation; Phosphoserine; Physiological; Positioning Attribute; Post-Translational Protein Processing; Protein Binding Domain; Protein Biosynthesis; 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; base; fitness; genome editing; human disease; mutant; new technology; new therapeutic target; novel; novel strategies; novel therapeutics; programs; 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.

项目摘要 健康且患病的生理状态受复杂的相互作用蛋白质的网络，赋予 在细胞中观察到的集体行为。这些蛋白质网络用翻译后修饰装饰 （PTM）确定其结构，功能和赋予细胞信号的特异性。磷酸化和 乙酰化代表了两个常见的PTM，这些PTM决定人类细胞中健康和疾病状态。例如， 14-3-3蛋白支架数千种重要的磷蛋白，有证据表明乙酰化可以 修改其功能。阐明PTM介导的信号网络的当前进展受到阻碍 通过研究细胞中瞬时PTM的挑战和有限的方法生产含有特定的蛋白质 改性氨基酸的组合。我们以前的努力利用了重新编码的大肠杆菌菌株（即在基因组上 重新编码的有机体）使用遗传密码扩展技术合成所有人类磷酸蛋白蛋白。 我们通过开发两种混合动力技术（名为HI-P）扩展了这项工作。 HI-P验证 先前观察到的磷酸化依赖性蛋白 - 蛋白质相互作用并确定了新的分数 在生化和 基于细胞的测定。我们的方法允许合成DNA输入以直接基于核糖体的磷蛋白 合成，因此创建了一种可编程的遗传工具来研究分子的人磷蛋白酶 等级。由于解密复杂蛋白网络需要研究多个PTM在孤立和 结合起来，预计拟议研究的关键贡献将扩大遗传学的能力 在14-3-3蛋白中的精确位置编码磷酸盐和乙酰化，以揭示PTM介导的蛋白质 - 蛋白质相互作用。具体目的：在此提案中，我们试图利用强大的技术基础， 专业知识和初步数据，可以用一个终止密码子（两个开放密码子）构建一个重新编码的大肠杆菌（AIM 1），开发一个能够同时编码磷酸化和乙酰化的蛋白质合成系统 氨基酸成蛋白质（AIM 2），并采用这些能力来否定PTM介导的14-3-3蛋白 网络交互（AIM 3）。意义：这项工作将是重要的，因为它将能够表达 可编程的人蛋白包含两个PTM，从而建立了一种新的方法来破译复合物 分子水平的人类信号网络。我们预计这项工作将阐明新颖的14-3-3蛋白 由PTM管辖的网络相互作用，并为生物分子和蛋白质机制提供了新的研究 可以用来开发针对人类疾病的新疗法。