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PLoS One. 2012;7(11):e50016. doi: 10.1371/journal.pone.0050016. Epub 2012 Nov 16.

A quantitative proteomic profile of the Nrf2-mediated antioxidant response of macrophages to oxidized LDL determined by multiplexed selected reaction monitoring.

Author information

1
Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America.

Abstract

The loading of macrophages with oxidized low density lipoprotein (LDL) is a key part of the initiation and progression of atherosclerosis. Oxidized LDL contains a wide ranging set of toxic species, yet the molecular events that allow macrophages to withstand loading with these toxic species are not completely characterized. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the cellular stress response. However, the specific parts of the Nrf2-dependent stress response are diverse, with both tissue- and treatment-dependent components. The goal of these experiments was to develop and use a quantitative proteomic approach to characterize the Nrf2-dependent response in macrophages to oxidized LDL. Cultured mouse macrophages, the J774 macrophage-like cell line, were treated with a combination of oxidized LDL, the Nrf2-stabilizing reagent tert- butylhydroquinone (tBHQ), and/or Nrf2 siRNA. Protein expression was determined using a quantitative proteomics assay based on selected reaction monitoring. The assay was multiplexed to monitor a set of 28 antioxidant and stress response proteins, 6 housekeeping proteins, and 1 non-endogenous standard protein. The results have two components. The first component is the validation of the multiplexed, quantitative proteomics assay. The assay is shown to be fundamentally quantitative, precise, and accurate. The second component is the characterization of the Nrf2-mediated stress response. Treatment with tBHQ and/or Nrf2 siRNA gave statistically significant changes in the expression of a subset of 11 proteins. Treatment with oxidized LDL gave statistically significant increases in the expression of 7 of those 11 proteins plus one additional protein. All of the oxLDL-mediated increases were attenuated by Nrf2 siRNA. These results reveal a specific, multifaceted response of the foam cells to the incoming toxic oxidized LDL.

PMID:
23166812
PMCID:
PMC3500347
DOI:
10.1371/journal.pone.0050016
[Indexed for MEDLINE]
Free PMC Article

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