Beavers William N, Rose Kristie L, Galligan James J, Mitchener Michelle M, Rouzer Carol A, Tallman Keri A, Lamberson Connor R, Wang Xiaojing, Hill Salisha, Ivanova Pavlina T, Brown H Alex, Zhang Bing, Porter Ned A, Marnett Lawrence J
A.B. Hancock Memorial Laboratory for Cancer Research, Departments of Chemistry, ‡Biochemistry, §Pharmacology, ∥Biomedical Informatics, ⊥Vanderbilt Mass Spectrometry Research Center, Vanderbilt Institute for Chemical Biology, Vanderbilt Center in Molecular Toxicology, Vanderbilt Ingram Cancer Center, Vanderbilt University , Nashville, Tennessee 37232, United States.
ACS Chem Biol. 2017 Aug 18;12(8):2062-2069. doi: 10.1021/acschembio.7b00480. Epub 2017 Jun 28.
Determining the impact of lipid electrophile-mediated protein damage that occurs during oxidative stress requires a comprehensive analysis of electrophile targets adducted under pathophysiological conditions. Incorporation of ω-alkynyl linoleic acid into the phospholipids of macrophages prior to activation by Kdo-lipid A, followed by protein extraction, click chemistry, and streptavidin affinity capture, enabled a systems-level survey of proteins adducted by lipid electrophiles generated endogenously during the inflammatory response. Results revealed a dramatic enrichment for membrane and mitochondrial proteins as targets for adduction. A marked decrease in adduction in the presence of MitoTEMPO demonstrated a primary role for mitochondrial superoxide in electrophile generation and indicated an important role for mitochondria as both a source and target of lipid electrophiles, a finding that has not been revealed by prior studies using exogenously provided electrophiles.
确定氧化应激期间发生的脂质亲电试剂介导的蛋白质损伤的影响，需要对病理生理条件下加成的亲电试剂靶点进行全面分析。在被Kdo-脂质A激活之前，将ω-炔基亚油酸掺入巨噬细胞的磷脂中，随后进行蛋白质提取、点击化学和链霉亲和素亲和捕获，从而能够对炎症反应期间内源性产生的脂质亲电试剂加成的蛋白质进行系统水平的调查。结果显示，膜蛋白和线粒体蛋白作为加成靶点显著富集。在存在MitoTEMPO的情况下，加成显著减少，这表明线粒体超氧化物在亲电试剂生成中起主要作用，并表明线粒体作为脂质亲电试剂的来源和靶点都具有重要作用，这一发现是之前使用外源性亲电试剂的研究所未揭示的。
