Defining Roles Of NitroTyrosine In Disease Via Genetic Code Expansion

 描述（由申请人提供）：通过使用3-硝基酪氨酸（nitroTyr）作为生物标志物，已经证明了活性氮物质在衰老以及80多种人类疾病（包括动脉粥样硬化、癌症、慢性疼痛、感染、神经变性和中风）中的作用。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。该提议的中心假设是硝基Tyr修饰的蛋白质是人类疾病中的关键参与者，并且理解它们在病理学中的机制作用将导致治疗干预的新机会。使用常规生物化学和基于细胞的方法的挑战是如何确定哪些硝基Tyr修饰在功能上是重要的，哪些是无关紧要的。PI已经表明，可以通过使用遗传密码扩增技术将硝基Tyr以靶向方式定量和位点特异性地掺入细菌中产生的重组蛋白中来克服这一障碍。这种方法现在已被用来提供前两个示范，即特定疾病中的特定硝基酪氨酸蛋白质具有改变的特性，这些特性暗示它们是病理学发展中的关键参与者。在一种情况下，硝化热休克蛋白90（Hsp 90）中的两个特定的酪氨酸中的任何一个可以导致肌萎缩侧索硬化症中的运动神经元死亡，在另一种情况下，硝化载脂蛋白A1蛋白中的单个Tyr导致其选择性掺入动脉粥样硬化斑块。促进确定硝基Tyr-蛋白的病理机制的下一步是能够在哺乳动物细胞中编码它们，使得可以直接确定硝基Tyr修饰如何在体内改变蛋白质功能、相互作用和调节。该提案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，以阐明酪氨酸硝化在已知生理重要性的生物学相关模型系统中改变蛋白质相互作用的机制。所选模型系统的中心是钙调蛋白（CaM）和Hsp 90的关键酪氨酸，以及它们的硝化如何改变钙对一氧化氮的调节和来自常见客户蛋白（内皮一氧化氮合酶）的超氧化物产生。所创建的工具将通过提供一种方法来评估哺乳动物细胞中任何给定蛋白质中特定硝基Tyr残基的功能影响，从而克服该领域的主要障碍。这项工作还将提供初步的见解，以开放的问题，如何硝化在特定的酪氨酸影响的选择功能， CaM和Hsp 90，以及酪氨酸硝化和磷酸化之间的相互作用。这项工作将通过提供一种全新的方法来产生持续的影响，该方法可用于了解酪氨酸硝化如何影响许多人类疾病的疾病进展。对于每一个发现硝基酪氨酸形成确实有助于病理发展的病例，该过程的绘制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.