A Quantitative Proteomic Profile of the Nrf2-Mediated Antioxidant Response of Macrophages to Oxidized LDL Determined by Multiplexed Selected Reaction Monitoring
A Quantitative Proteomic Profile of the Nrf2-Mediated Antioxidant Response of Macrophages to Oxidized LDL Determined by Multiplexed Selected Reaction Monitoring
复制标题
DOI：
10.1371/journal.pone.0050016
复制
发表时间：
2012-11-16
期刊：
影响因子：
3.7
通讯作者：
Kinter, Michael
中科院分区：
文献类型：
--
作者：
Kinter, Caroline S.;Lundie, Jillian M.;Kinter, Michael
巨噬细胞负载氧化低密度脂蛋白（LDL）是动脉粥样硬化发生和进展的关键部分。氧化低密度脂蛋白含有多种有毒物质，但巨噬细胞能够承受这些有毒物质的分子事件尚未完全表征。转录因子核因子（红细胞衍生 2）样 2 (Nrf2) 是细胞应激反应的主要调节因子。然而，Nrf2 依赖性应激反应的具体部分是多种多样的，既有组织依赖性的成分，也有治疗依赖性的成分。这些实验的目标是开发和使用定量蛋白质组学方法来表征巨噬细胞对氧化 LDL 的 Nrf2 依赖性反应。培养的小鼠巨噬细胞（J774 巨噬细胞样细胞系）用氧化 LDL、Nrf2 稳定试剂叔丁基氢醌 (tBHQ) 和/或 Nrf2 siRNA 的组合进行处理。使用基于选定反应监测的定量蛋白质组学测定来确定蛋白质表达。该检测多重检测以监测一组 28 种抗氧化剂和应激反应蛋白、6 种管家蛋白和 1 种非内源标准蛋白。结果有两个组成部分。第一个组成部分是多重定量蛋白质组学测定的验证。该测定法基本上是定量的、精确的和准确的。第二个部分是 Nrf2 介导的应激反应的表征。使用 tBHQ 和/或 Nrf2 siRNA 治疗后，11 种蛋白质的子集的表达发生了统计学上的显着变化。使用氧化 LDL 进行治疗后，这 11 种蛋白质中的 7 种以及另外一种蛋白质的表达显着增加。所有 oxLDL 介导的增加均被 Nrf2 siRNA 减弱。这些结果揭示了泡沫细胞对进入的有毒氧化低密度脂蛋白的特定的、多方面的反应。
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.