A novel approach to define the roles of oxidative and nitrative post-trasnlational modifications of tryptophan in human biology

一种定义色氨酸氧化和硝基翻译后修饰在人类生物学中的作用的新方法

• 批准号: 9368167
• 负责人: Abhishek Chatterjee
• 金额: $ 30.52万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9368167
• 关键词: Affinity; Amino Acids; Amino Acyl-tRNA Synthetases; Atherosclerosis; Binding; Biological; Biology; Charge; Cysteine; Development; Disease; Engineering; Escherichia coli; Eukaryota; Eukaryotic Cell; Genetic Code; Half-Life; Health; Human; Human Biology; In Vitro; Inflammation; Invaded; Knowledge; Kynurenine; Ligands; Mammalian Cell; Mass Spectrum Analysis; Metabolic; Metabolism; Modification; Molecular; N' -formylkynurenine; Nerve Degeneration; Nitrates; Nitrogen; Oxidation-Reduction; Oxides; Oxygen; Pathogenesis; Phosphoglycerate Kinase; Physiological; Play; Post-Translational Protein Processing; Process; Production; Property; Proteins; Proteome; Proteomics; Reagent; Reporting; Role; Signal Transduction; Site; Stress; Stroke; System; Technology; Therapeutic Intervention; Time; Tryptophan; Tryptophan-tRNA Ligase; Tyrosine; Variant; Work; base; enolase; enzyme activity; human disease; in vivo; insight; nitration; novel; novel strategies; oxidation; pathogen; protein expression; protein folding; protein function; protein protein interaction; protein structure; technology development; tryptophan analog; unnatural amino acids

Project Summary Regulated production of reactive oxygen and nitrogen species serves important roles in our biology, such as providing unique mechanisms for cell signaling, as well as defense against invading pathogens. However, their elevated levels are associated with numerous human diseases including atherosclerosis, stroke, neurodegeneration, inflammation, etc. These reactive agents modify redox-active amino acid residues on proteins, causing structural and functional changes that serve as the molecular basis for signaling, as well as the pathogenesis of numerous diseases. Several oxidative and nitrative post- translational modifications (PTMs) of tryptophan have been recently identified in many sites of our proteome. These modifications are not distributed randomly; rather, specific tryptophan residues on specific proteins are found to be selectively modified. While a limited number of reports have shown that these modifications can trigger alterations in protein structure and function, physiological consequences of the tryptophan modifications observed in our proteome remains mostly unclear. At the core of this knowledge gap lies our current inability to generate target proteins in a homogeneously modified form, and ask how their properties are altered in vivo and in vitro. To overcome this limitation, here we propose the development of technology that will enable co-translational site-specific incorporation of physiologically relevant modified tryptophan residues into any target protein. We have recently developed a unique tryptophanyl-tRNA synthetase (TrpRS)/tRNATrp pair that can be used to site-specifically incorporate unnatural amino acids into proteins expressed in both E. coli and eukaryotic cells. We have further demonstrated our ability to engineer this TrpRS/tRNATrp pair to enable site-specific incorporation of a variety of tryptophan analogs into proteins. Here we propose further development of this platform to allow co-translational site-specific incorporation of physiologically relevant oxidized/nitrated tryptophan derivatives, which will for the first time enable facile expression of target proteins harboring these modified tryptophans at predefined sites in both E. coli as well as mammalian cells. We will further use this platform to investigate the role of tryptophan nitration using two established targets, phosphoglycerate kinase 1 and α-enolase, both important human metabolic proteins. Our work will establish a novel and general approach for understanding the role of oxidative/nitrative modifications of tryptophan residues in human health and disease. The ability to characterize the elusive connections between oxidative/nitrative tryptophan modification and various human diseases will also uncover new opportunities for therapeutic intervention.

项目摘要 调节活性氧和氮的产生在我们的生物学中起着重要作用， 为细胞信号传导提供独特的机制，以及防御入侵的病原体。 然而，它们的水平升高与许多人类疾病相关，包括动脉粥样硬化， 中风、神经变性、炎症等。这些反应剂修饰氧化还原活性氨基酸 蛋白质上的残基，引起结构和功能的变化，作为分子基础， 信号传导，以及许多疾病的发病机制。几种氧化和硝化后- 色氨酸的翻译修饰（PTM）最近已经在我们的许多位点被发现。 蛋白质组这些修饰不是随机分布的;相反，表面上的特定色氨酸残基 发现特异性蛋白质被选择性修饰。虽然数量有限的报告表明， 这些修饰可以引发蛋白质结构和功能的改变， 在我们的蛋白质组中观察到的色氨酸修饰大多仍不清楚。在这个问题的核心 知识差距在于我们目前无法以均匀修饰的形式产生靶蛋白， 询问它们的特性在体内和体外是如何改变的。为了克服这一限制，我们在这里提出了 开发能够使生理学上的共翻译位点特异性掺入的技术 相关修饰的色氨酸残基转化为任何靶蛋白。我们最近开发了一种独特的 可用于位点特异性掺入的双氢酰-tRNA合成酶（TrpRS）/tRNATrp对 将非天然氨基酸转化为在E.大肠杆菌和真核细胞。我们进一步 证明了我们有能力工程化这种TrpRS/tRNATrp对，使位点特异性掺入的一个 各种色氨酸类似物转化为蛋白质。在这里，我们建议进一步发展这个平台， 生理学相关的氧化/硝化色氨酸的共翻译位点特异性掺入 衍生物，这将首次使携带这些修饰的靶蛋白的容易表达成为可能。 在E.大肠杆菌以及哺乳动物细胞。我们将进一步利用这个平台 为了研究色氨酸硝化的作用，使用两个已建立的靶点，磷酸甘油酸激酶1和 α-烯醇化酶是人体重要的代谢蛋白。我们的工作将建立一个新的和一般的 色氨酸残基的氧化/硝化修饰在人体内的作用 健康和疾病。描述氧化/硝化之间难以捉摸的联系的能力 色氨酸修饰和各种人类疾病也将揭示新的治疗机会， 干预