Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
Chem Res Toxicol. 2013 Feb 18;26(2):270-9. doi: 10.1021/tx300449g. Epub 2012 Dec 24.
Oxidation of membrane phospholipids is associated with inflammation, neurodegenerative disease, and cancer. Oxyradical damage to phospholipids results in the production of reactive aldehydes that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we demonstrate that peptidyl-prolyl cis/trans-isomerase A1 (Pin1), an enzyme that catalyzes the conversion of the peptide bond of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry resulted in detection of Michael adducts at the active site residues His-157 and Cys-113. Time and concentration dependencies indicate that Cys-113 is the primary site of HNE modification. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and Western blotting with anti-Pin1 antibody. Furthermore, orbitrap MS data support the adduction of Cys-113 in the Pin1 active site upon HNE treatment of MDA-MB-231 cells. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation.
膜磷脂的氧化与炎症、神经退行性疾病和癌症有关。氧自由基对磷脂的损伤导致反应性醛的产生，这些醛与蛋白质结合并调节其功能。4-羟基壬烯醛（HNE）是脂质氧化的常见产物，与暴露的 Cys、His 或 Lys 残基上的蛋白质结合。在这里，我们证明了肽酰脯氨酰顺/反异构酶 A1（Pin1），一种催化信号蛋白中 pSer/pThr-Pro 部分肽键从顺式到反式转化的酶，极易受到 HNE 修饰。用 HNE 孵育纯化的 Pin1 ，然后用 MALDI-TOF/TOF 质谱法检测到活性位点残基 His-157 和 Cys-113 的 Michael 加合物。时间和浓度依赖性表明 Cys-113 是 HNE 修饰的主要部位。MDA-MB-231 乳腺癌细胞用 8-炔基-HNE 处理后，通过点击化学与生物素缀合，然后用链霉亲和素下拉和抗 Pin1 抗体进行 Western blot 检测，判断 Pin1 被加合物修饰。此外，轨道阱 MS 数据支持 MDA-MB-231 细胞中 HNE 处理后 Pin1 活性位点的 Cys-113 加合物。MDA-MB-231 细胞中 Pin1 的 siRNA 敲低部分保护细胞免受 HNE 诱导的毒性。最近的研究表明，Pin1 是绿茶多酚化学预防作用的重要分子靶点。本研究确立了它也是脂质过氧化产物亲电修饰的靶标。
