Defining Roles Of NitroTyrosine In Disease Via Genetic Code Expansion

 描述(申请人提供)：通过使用3-硝基酪氨酸(Nitrotyr)作为生物标记物，证明了活性氮物种在衰老以及80多种人类疾病中的作用，包括动脉粥样硬化、癌症、慢性疼痛、感染、神经变性和中风。在这些条件下，酪氨酸硝化不是随机分布的，但某些蛋白质上的特定酪氨酸更容易被修饰。这一提议的中心假设是，硝基酪氨酸修饰的蛋白质是人类疾病的关键参与者，了解它们在病理中的机制作用将为治疗干预带来新的机会。使用传统的生化和基于细胞的方法面临的挑战是如何确定哪些硝基酪氨酸修饰在功能上是重要的，哪些是无关紧要的。PI已经表明，通过使用遗传密码扩展技术，以有针对性的方式将硝基酪氨酸定量和定点地整合到细菌生产的重组蛋白中，可以克服这一障碍。这种方法现在已经被用来提供前两个证明，在给定的疾病中特定的硝基酪氨酸蛋白改变了属性，这意味着它们在病理学的发展中扮演着关键角色。在一个案例中，热休克蛋白90(Hsp90)中两个特定酪氨酸的硝化可导致肌萎缩侧索硬化症中运动神经元的死亡，而在另一个案例中，载脂蛋白A1中单个Tyr的硝化导致其选择性地整合到动脉粥样硬化斑块中。为了便于确定硝基酪氨酸蛋白的病理机制，下一步是能够在哺乳动物细胞中对其进行编码，以便在体内直接确定硝基酪氨酸修饰如何改变蛋白质的功能、相互作用和调节。这项建议的重点是追求两个目标，包括(1)开发哺乳动物表达硝基酪氨酸蛋白所需的工具，以及(2)应用这些工具进行体外和体内研究，以阐明酪氨酸硝化改变已知生理重要性的生物相关模型系统中蛋白质相互作用的机制。选定的模型系统以钙调蛋白(CaM)和Hsp90的关键酪氨酸为中心，以及它们的硝化作用如何改变钙对一氧化氮的调节，以及从常见的客户蛋白-内皮型一氧化氮合酶-产生超氧化物的过程。创建的工具将克服该领域的主要障碍，因为它提供了一种方法，在哺乳动物细胞中评估任何给定蛋白质中特定的硝基酪氨酸残基的功能影响。这项工作还将对特定酪氨酸的硝化作用如何影响特定酪氨酸的某些功能的未决问题提供初步的见解 CaM和Hsp90，以及酪氨酸硝化和磷酸化之间的相互作用。这项工作将产生持久的影响，因为它提供了一种全新的方法，可以用来理解酪氨酸硝化如何影响它发生的许多人类疾病的疾病进展。对于每一个被发现硝基酪氨酸形成确实有助于病理发展的病例，这一过程的图谱将为治疗干预开辟一条新的途径。

 DESCRIPTION (provided by applicant): A role for reactive nitrogen species in aging as well as in over eighty human diseases including atherosclerosis, cancer, chronic pain, infection, neurodegeneration, and stroke has been demonstrated by using 3-nitrotyrosine (nitroTyr) as a biomarker. In these conditions, tyrosine nitration is not randomly distributed, but specific tyrosines on certain proteins are more readily modified. The central hypothesis of this proposal is that nitroTyr- modified proteins are key players in human disease and that understanding their mechanistic role in pathology will lead to new opportunities for therapeutic intervention. The challenge using conventional biochemical and cell-based approaches has been how to determine which nitroTyr modifications are functionally significant and which are inconsequential. The PI has shown that this hurdle can be overcome by using genetic code expansion technology to quantitatively and site-specifically incorporate nitroTyr in a targeted manner into recombinant proteins produced in bacteria. This approach has now been used to provide the first two demonstrations that specific nitroTyr-proteins in a given disease have altered properties that implicate them as key players in the development of pathology. In one case, the nitration of either of two specific tyrosines in heat shock protein 90 (Hsp90) can cause motor neuron death in amyotrophic lateral sclerosis, and in the other case that the nitration of a single Tyr in the protein Apolipoprotein A1 leads to its selective incorporation into atherosclerotc plaques. The next step in facilitating determination of the mechanisms of pathology for nitroTyr-proteins is to be able to encode them in mammalian cells so that one can directly determine in vivo how nitroTyr modifications alter protein function, interactions, and regulation. The focus of this proposal is to pursue two aims that encompass (1) developing the needed tools for mammalian expression of nitroTyr-proteins, and (2) applying the tools to carry out both in vitro and in vivo studies to elucidate the mechanisms by which tyrosine nitration alters protein interactions in a biologically relevant model system of known physiological importance. The selected model system centers on key tyrosines of calmodulin (CaM) and Hsp90, and how their nitration alters calcium regulation of nitric oxide and superoxide production from a common client protein, the endothelial nitric oxide synthase. The tools created will overcome a major roadblock in the field by providing an approach to assess in mammalian cells the functional impacts of specific nitroTyr residues in any given protein. The work will also provide initial insights into the open questions of how nitration at specific tyrosines impacts select functions of CaM and Hsp90, and the interplay between tyrosine nitration and phosphorylation. This work will have a sustained impact by providing a fundamentally new approach that can be used to understand how tyrosine nitration affects disease progression in the many human diseases in which it occurs. And for every case in which it is discovered that nitroTyr formation does contribute to pathology development, the mapping of that process will open up a new avenue for therapeutic intervention.