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.
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