Protein Modification with Oxidative Stress in ALI

ALI 中氧化应激的蛋白质修饰

• 批准号: 7796691
• 负责人: DAVID W. SPEICHER
• 金额: $ 35.67万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7796691
• 关键词: Active Sites; Acute Lung Injury; Antioxidants; Arts; Biochemical; Biological Markers; Blood; Blood Proteins; Cells; Cohort Studies; Collaborations; Development; Diagnostic; Diagnostic tests; Enzymatic Biochemistry; Enzyme Activation; Enzymes; Experimental Models; Goals; Human; Hyperoxia; Immunoassay; Lipids; Lung; Mass Spectrum Analysis; Methods; Modeling; Modification; Molecular; Mus; Nitrates; Oxidative Stress; Pathway interactions; Patients; Pattern; Plasma; Plasma Proteins; Play; Post-Translational Protein Processing; Process; Property; Proteins; Proteomics; Reactive Oxygen Species; Recombinant Proteins; Recombinants; Resources; Role; Sampling; Serological; Structure; Structure of parenchyma of lung; Structure-Activity Relationship; Substrate Interaction; Testing; Thermodynamics; Tissues; Trauma; Validation; analytical ultracentrifugation; antioxidant therapy; base; biosignature; cohort; in vivo; insight; lung injury; macromolecular assembly; minimally invasive; mouse model; mutant; nanocarrier; novel; outcome forecast; oxidation; oxidative damage; peroxiredoxin; prognostic; protein profiling; sedimentation velocity; self assembly

We will employ state-of-the-art biochemical, biophysical, and proteomic approaches to explore the role of oxidative stress in acute lung injury (ALI). The overall goals are to: 1) define the mechanism of peroxiredoxin 6's (Prdx 6) antioxidant activity and its role in protection from oxidative stress; 2) gain novel insights into the molecular mechanism(s) of lung damage caused by oxidative stress by identifying critical lung proteins that are oxidatively modified under different experimental conditions in mouse models, and 3) identify human plasma biomarkers of lung injury and ALI development. A major focus (Aim 1) will be to investigate structure-function relationships of Prdx 6 in close collaboration. Specifically, we will use analytical ultracentrifugation, mass spectrometry, and related biochemical and biophysical approaches to characterize recombinant Prdx 6 self-assembly and interaction with wild type piGST and a piGST mutant. In parallel with these biophysical/structural studies, we will examine functional properties of Prdx 6, particularly substrate interactions and enzymology. Aim 1 will also evaluate oxidative modifications on Prdx 6 isolated from mouse lungs using the models of oxidative stress being studied. In related studies, Aim 2 will systematically identify other lung proteins that are oxidatively modified and evaluate changes in these modifications in mice under normoxic and hyperoxic conditions when Prdx 6 is expressed at normal levels, over-expressed, or underexpressed. The ability of nanocarrier anti-oxidant therapy to minimize oxidative damage of proteins after oxidative stress will also be assessed. In summary, Aims 1 and 2 will test the hypothesis that Prdx 6 plays a central role in protecting lung tissue from excessive oxidative damage under hyperoxic conditions. In addition, Aim 3 will test the related hypothesis that oxidative stress resulting from severe trauma in patients and the resulting lung tissue damage will induce changes in blood protein profiles that can form the basis for minimally invasive diagnostic tests of ALI. This aim will utilize a unique resource of plasma samples from patients in a trauma cohort study of ALI/ARDS. Two complementary novel proteomic methods will be used to identify novel biomarkers or biosignatures of ALI in these human plasma samples.

我们将采用最先进的生物化学，生物物理学和蛋白质组学方法来探讨氧化应激在急性肺损伤（ALI）中的作用。总体目标是：1）定义过氧化物氧还蛋白6（Prdx 6）抗氧化活性的机制及其在保护免受氧化应激中的作用; 2）通过在小鼠模型中鉴定在不同实验条件下被氧化修饰的关键肺蛋白，获得对氧化应激引起的肺损伤的分子机制的新见解; 3）鉴定肺损伤和ALI发展的人血浆生物标志物。一个主要重点（目标1）将是密切合作，研究Prdx 6的结构-功能关系。具体而言，我们将使用分析超离心，质谱，和相关的生化和生物物理方法来表征重组Prdx 6自组装和与野生型piGST和piGST突变体的相互作用。在进行这些生物物理/结构研究的同时，我们将研究Prdx 6的功能特性，特别是底物相互作用和酶学。目的1还将使用正在研究的氧化应激模型评价从小鼠肺分离的Prdx 6的氧化修饰。在相关研究中，Aim 2将系统地鉴定其他被氧化修饰的肺蛋白，并评估当Prdx 6以正常水平表达、过表达或低表达时，在常氧和高氧条件下小鼠中这些修饰的变化。纳米载体抗氧化治疗最大限度地减少氧化损伤的能力 还将评估氧化应激后的蛋白质。总之，目的1和2将检验Prdx 6在高氧条件下保护肺组织免受过度氧化损伤中起核心作用的假设。此外，目标3将测试相关假设，即患者严重创伤引起的氧化应激和由此产生的肺组织损伤将引起血液蛋白谱的变化，这些变化可以构成ALI微创诊断测试的基础。这一目标将在ALI/ARDS的创伤队列研究中利用患者的独特血浆样本资源。两种互补的新型蛋白质组学方法将用于鉴定这些人血浆样本中ALI的新型生物标志物或生物特征。