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（适用提供）：反应性氮种在衰老以及八十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，神经退行性变性和中风，通过使用3-硝基酪氨酸（Nitrotyr）作为生物标志物。在这种情况下，酪氨酸硝化不是随机分布的，但是某些蛋白质上的特定酪氨酸更容易修饰。该提议的核心假设是，硝化蛋白质是人类疾病的关键参与者，并且了解其在病理学中的机理作用将为治疗干预带来新的机会。使用常规生化和基于细胞的方法的挑战是如何确定哪些硝化含量修饰在功能上是显着的，哪些是无关紧要的。 PI表明，可以通过使用遗传密码扩展技术以靶向方式将硝化硝化物以靶向方式掺入细菌中产生的重组蛋白中，并特异性地掺入硝化硝酸盐。现在，这种方法已被用来提供前两个证明，即给定疾病中特定的硝化蛋白质蛋白改变了特性，使它们成为病理发展的关键参与者。在一种情况下，热激蛋白90（HSP90）中两种特异性酪氨酸中的任何一个都可能导致肌萎缩性后来的硬化症中的运动神经元死亡，而在另一种情况下，单个TYR在蛋白质载脂蛋白A1中的硝化作用是将其精选掺入蛋白质载脂蛋白A1中的硝化作用。支持确定硝酸蛋白质病理机理的下一步是能够在哺乳动物细胞中编码它们，以便可以直接确定体内硝基核修饰如何改变蛋白质功能，相互作用和调节。该提案的重点是追求两个目标，其中包括（1）开发哺乳动物表达硝化酚蛋白的所需工具，以及（2）应用工具在体外和体内研究中同时进行体外和体内研究，以阐明酪氨酸硝化物在生物学上相关模型中的蛋白质相互作用的机制，从而使蛋白质相互作用具有相关的已知物理模型。选定的模型系统集中于钙调蛋白（CAM）和HSP90的关键酪氨酸，以及它们的硝化如何从普通客户蛋白（内皮一氧化氮合酶）中改变一氧化氮和超氧化物的钙调节。创建的工具将通过提供一种评估哺乳动物细胞的方法来克服该领域的主要障碍。这项工作还将为开放问题提供有关特定酪氨酸如何影响的选择功能的开放问题的初步见解。 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从根本上的新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疾病进展。对于每种发现Nitrotyr组的形成确实有助于病理发展的情况，该过程的映射将为治疗干预提供新的途径。