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

项目摘要 健康和患病的生理状态由相互作用的蛋白质组成的复杂网络控制，这些蛋白质赋予 在细胞中观察到的集体行为。这些蛋白质网络被翻译后修饰修饰。 (PTM)决定它们的结构、功能，并赋予细胞信号的特异性。磷酸化和 乙酰化代表两种常见的PTM，它们决定了人类细胞的健康和疾病状态。例如, 14-3-3蛋白是数千种重要的磷酸蛋白的骨架，有证据表明乙酰化可以 修改其功能。目前对PTM介导的信号网络的阐明进展受阻 通过在细胞中研究瞬时PTMS的挑战和有限的方法来生产含有特定蛋白质的 经修饰的氨基酸的组合。我们之前的工作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来合成所有的人类磷酸丝氨酸蛋白。 我们通过开发一种名为HI-P的类似双杂交的技术来扩展这项工作。Hi-P验证 先前观察到的依赖磷酸化的蛋白质-蛋白质相互作用和识别的数十个新的 磷酸丝氨酸介导的跨人类蛋白质组的相互作用已在生化和 基于细胞的分析。我们的方法允许合成DNA输入直接基于核糖体的磷酸蛋白 合成，从而创造了一种可编程的遗传工具来在分子水平上研究人类磷酸蛋白质组 水平。由于破译复杂的蛋白质网络需要研究多个PTM在分离和 总而言之，这项拟议研究的关键贡献有望扩大遗传研究的能力 编码14-3-3蛋白中精确位置的磷酸丝氨酸和乙酰化，揭示PTM介导的蛋白- 蛋白质的相互作用。具体目标：在这项提案中，我们寻求利用强大的技术基础， 专业知识和初步数据，构建具有单一终止密码子(两个开放密码子)的重新编码的大肠杆菌(AIM 1)，开发一种可同时编码磷酸化和乙酰化的蛋白质合成系统 氨基酸转化为蛋白质(目标2)，并利用这些能力解开PTM介导的14-3-3蛋白 网络互动(目标3)。意义：这项工作将具有重要意义，因为它将使 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体的新方法 人类在分子水平上的信号网络。我们预计这项工作将阐明新的14-3-3蛋白 由PTMS控制的网络相互作用，使对生物分子和蛋白质机制的新研究成为可能 这可以用来开发治疗人类疾病的新疗法。

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.