Protein Modification with Oxidative Stress in ALI

我们将使用最先进的生化、生物物理和蛋白质组学方法来探索氧化应激在急性肺损伤(ALI)中的作用。总体目标是：1)确定过氧化还蛋白6‘S(PRDX 6)的抗氧化活性及其在抗氧化应激保护中的作用；2)通过在小鼠模型中识别在不同实验条件下被氧化修饰的关键肺蛋白质，以获得对氧化应激导致肺损伤的分子机制(S)的新见解，以及3)确定肺损伤和ALI发生的人类血浆生物标志物。一个主要的焦点(目标1)将是在密切合作下研究PRDX 6的结构-功能关系。具体地说，我们将使用分析超速离心法、质谱仪以及相关的生化和生物物理方法来表征重组PRDX 6的自组装以及与野生型piGST和一个piGST突变体的相互作用。在这些生物物理/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。AIM 1还将使用正在研究的氧化应激模型评估从小鼠肺中分离出的PRDX 6的氧化修饰。在相关研究中，AIM 2将系统地识别其他被氧化修饰的肺蛋白质，并评估当PRDX 6在正常水平、过度表达或低表达时，在常氧和高氧条件下小鼠这些修饰的变化。纳米载体抗氧化剂治疗对心肌细胞氧化损伤的影响 氧化应激后的蛋白质也将被评估。总之，AIMS 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发挥核心作用的假设。此外，AIM 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导致血液蛋白质谱变化，这可能构成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独特的血浆样本来源。两种互补的新蛋白质组学方法将用于在这些人血浆样本中识别ALI的新生物标记物或生物标志。

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.