Protein Modification with Oxidative Stress in ALI

我们将采用最先进的生化、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)明确过氧化物还氧蛋白6 （Prdx 6）抗氧化活性的机制及其在氧化应激中的保护作用；2)通过鉴定小鼠模型在不同实验条件下氧化修饰的关键肺蛋白，获得氧化应激引起肺损伤的分子机制的新见解；3)鉴定肺损伤和ALI发展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。具体来说，我们将使用分析性超离心、质谱和相关的生化和生物物理方法来表征重组Prdx 6的自组装以及与野生型piGST和piGST突变体的相互作用。与这些生物物理/结构研究并行，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还将利用正在研究的氧化应激模型评估从小鼠肺中分离的Prdx 6的氧化修饰。在相关研究中，Aim 2将系统地鉴定氧化修饰的其他肺蛋白，并评估在常氧和高氧条件下，当Prdx 6在正常水平、过表达或低表达时，这些修饰在小鼠中的变化。纳米载体抗氧化治疗的能力，以尽量减少氧化损伤

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.