Defining Roles Of NitroTyrosine In Disease Via Genetic Code Expansion

通过遗传密码扩展定义硝基酪氨酸在疾病中的作用

基本信息

批准号：
9105425
负责人：
RYAN A MEHL
金额：
$ 27.27万
依托单位：
OREGON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-05 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9105425
关键词：
3-nitrotyrosine Affect Aging Alzheimer&apos s Disease Amino Acyl-tRNA Synthetases Amyotrophic Lateral Sclerosis Apolipoprotein A-I Arthritis Atherosclerosis Bacteria Biochemical Biological Markers Biological Models Calcium Calmodulin Cells Chronic Cancer Pain Client Collaborations Complex Development Disease Disease Progression Escherichia Escherichia coli Generations Genetic Code Health Heat-Shock Proteins 90 Human In Situ In Vitro Infection Inflammation Lead Mammalian Cell Maps Mediating Metabolic Methods Modification Monitor Motor Neurons NOS3 gene Nerve Degeneration Nitrates Nitric Oxide Organ Oxidative Stress Parkinson Disease Pathology Phosphorylation Physiological Play Post-Translational Protein Processing Predisposition Process Production Property Protein Tyrosine Kinase Proteins Reactive Nitrogen Species Recombinant Proteins Recombinants Regulation Reporter Research Role Sepsis Severities Site Stroke Superoxides Systems Development Technology Testing Therapeutic Intervention Tissues Translations Tyrosine Work Xenograft procedure analog base cell injury experience human disease in vivo insight link protein lung injury neuron loss nitration novel strategies protein expression protein function technology development tool

项目摘要

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.

描述（由申请人提供）：通过使用 3-硝基酪氨酸（硝基Tyr）作为活性氮，已证明活性氮在衰老以及 80 多种人类疾病（包括动脉粥样硬化、癌症、慢性疼痛、感染、神经退行性变和中风）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酪氨酸被修饰。蛋白质是人类疾病的关键参与者，了解它们在病理学中的机制作用将为治疗干预带来新的机会，使用传统的生化和细胞方法面临的挑战是如何确定哪些硝基酪氨酸修饰具有功能意义，哪些修饰无关紧要。 PI 表明，可以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地、位点特异性地整合到细菌产生的重组蛋白中来克服这一障碍。这种方法现已用于提供前两种方法。证明特定疾病中的特定硝基酪氨酸已经改变了特性，这意味着它们在病理学发展中发挥着关键作用。在一种情况下，热休克蛋白 90 (Hsp90) 中两个特定酪氨酸之一的硝化可导致运动神经元死亡。在肌萎缩侧索硬化症中，在另一种情况下，载脂蛋白 A1 中单个 Tyr 的硝化导致其选择性掺入动脉粥样硬化中促进确定硝基酪氨酸蛋白病理机制的下一步是能够在哺乳动物细胞中对其进行编码，以便可以在体内直接确定硝基酪氨酸修饰如何改变蛋白质功能、相互作用和调节。该提案旨在实现两个目标，包括（1）开发哺乳动物表达硝基酪蛋白所需的工具，以及（2）应用这些工具进行体外和体内研究，以阐明其机制酪氨酸硝化改变具有已知生理重要性的生物相关模型系统中的蛋白质相互作用。所选模型系统以钙调蛋白 (CaM) 和 Hsp90 的关键酪氨酸为中心，以及它们的硝化如何改变常见客户蛋白对一氧化氮和超氧化物产生的钙调节。内皮一氧化氮合酶所创建的工具将通过提供一种评估哺乳动物细胞中特定功能影响的方法来克服该领域的主要障碍。这项工作还将为特定酪氨酸的硝化如何影响特定功能的开放性问题提供初步见解。 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其发生的许多人类疾病的进展。在每一个发现硝基酪氨酸形成确实有助于病理发展的病例中，对该过程的绘制将为治疗干预开辟一条新途径。