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

项目摘要 活性氧和氮物种的调节产生在我们的生物学中扮演着重要的角色，例如 因为提供了独特的细胞信号传递机制，以及对入侵病原体的防御。 然而，它们的水平升高与许多人类疾病有关，包括动脉粥样硬化， 中风、神经退行性变、炎症等。这些反应物修改氧化还原活性氨基酸 蛋白质上的残基，导致结构和功能的变化，作为分子基础 信号，以及许多疾病的发病机制。几个氧化和硝化后- 色氨酸的翻译修饰(PTM)最近在我们的许多部位被发现 蛋白质组。这些修饰不是随机分布的；相反，特定的色氨酸残基在 特定的蛋白质被发现被选择性地修饰。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，生理后果 在我们的蛋白质组中观察到的色氨酸修饰大部分仍不清楚。在这件事的核心 知识鸿沟在于我们目前无法以均一修饰的形式产生靶蛋白，并且 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，我们在这里建议使用 开发技术，使生理学上特定部位的共翻译结合成为可能 将相关修饰的色氨酸残基转化为任何目标蛋白。我们最近开发了一种独特的 色氨酰-tRNA合成酶(TrpRS)/tRNATrp对，可用于定点整合 将非天然氨基酸转化为在大肠杆菌和真核细胞中表达的蛋白质。我们还有更多 展示了我们设计TrpRS/tRNATrp对的能力，以实现特定于站点的整合 各种色氨酸类似物变成蛋白质。在此，我们建议进一步开发此平台，以允许 生理上相关的氧化/硝化色氨酸的共翻译位点特异性掺入 衍生物，这将首次使含有这些修饰的靶蛋白的表达变得容易 色氨酸在大肠杆菌和哺乳动物细胞中的预定义位置。我们将进一步使用这个平台 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，两种重要的人体代谢蛋白。我们的工作将建立一个新颖而普遍的 了解色氨酸残基的氧化/硝化修饰在人体内作用的方法 健康和疾病。能够描述氧化/硝化之间难以捉摸的联系 色氨酸修饰和各种人类疾病也将发现新的治疗机会 干预。

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.