Kobeissy Firas H, Guingab-Cagmat Joy, Bruijnzeel Adriaan W, Gold Mark S, Wang Kevin
Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.
Department of Psychiatry, Center for Neuroproteomics and Biomarkers Research, University of Florida, Gainesville, FL, USA.
Methods Mol Biol. 2017;1598:353-372. doi: 10.1007/978-1-4939-6952-4_18.
Second-hand smoke (SHS) exposure leads to the death of approximately 48,000 nonsmokers per year in the United States alone. SHS exposure has been associated with cardiovascular, respiratory, and neurodegenerative diseases. While cardiac function abnormalities and lung cancer due to SHS have been well characterized, brain injury due to SHS has not undergone a full systematic evaluation. Oxidative stress and nitration have been associated with smoking and SHS exposure. Animal studies suggest that exposure to tobacco smoke increases oxidative stress. Oxidative stress is characterized by an increase in reactive oxygen and nitrogen species (ROS/RNS). Among the oxidative mechanisms affecting protein functionality is the posttranslational modification (PTM)-mediated tyrosine nitration. Protein tyrosine nitration, a covalent posttranslational modification, is commonly used as a marker of cellular oxidative stress associated with the pathogenesis of several neurodegenerative diseases. In our previous published work, the utility of a targeted proteomic approach has been evaluated to identify two brain abundant proteins in an in vivo SHS rat model namely the GAPDH and UCH-L1. In this current study, mass spectrometric-based proteomic and complementary biochemical methods were used to characterize the SHS-induced brain nitroproteome followed by bioinformatics/systems biology approach analysis to characterize protein interaction map. Sprague Dawley rats were exposed to SHS for 5 weeks and then cortical tissues were collected. Nitroprotein enrichment was performed via 3-Nitro tyrosine (3-NT) immunoprecipitation of brain lysates proteins. Protein nitration was validated via Western blotting to confirm the presence of nitroproteins complemented by gel-free neuroproteomic analysis by data-dependent LC-MS/MS. We identified 29 differentially expressed proteins in the 3-NT-enriched samples; seven of these proteins were unique to SHS exposure. Network analysis revealed an association of the proteins to different cellular processes including oxidative stress, ROS generation, and cell death-related pathway. This confirms the association of oxidative stress mechanisms with SHS which may contribute to neuronal injury, an area that has not been well studied in the area smoking.
仅在美国，每年就有大约48000名不吸烟者因接触二手烟（SHS）而死亡。接触二手烟与心血管疾病、呼吸系统疾病和神经退行性疾病有关。虽然由于接触二手烟导致的心脏功能异常和肺癌已得到充分表征，但由于接触二手烟导致的脑损伤尚未进行全面系统评估。氧化应激和硝化作用与吸烟及接触二手烟有关。动物研究表明，接触烟草烟雾会增加氧化应激。氧化应激的特征是活性氧和氮物种（ROS/RNS）增加。影响蛋白质功能的氧化机制之一是翻译后修饰（PTM）介导的酪氨酸硝化。蛋白质酪氨酸硝化是一种共价翻译后修饰，通常用作与几种神经退行性疾病发病机制相关的细胞氧化应激标志物。在我们之前发表的工作中，已经评估了靶向蛋白质组学方法在体内SHS大鼠模型中鉴定两种脑丰度蛋白（即甘油醛-3-磷酸脱氢酶（GAPDH）和泛素羧基末端水解酶L1（UCH-L1））的效用。在本研究中，使用基于质谱的蛋白质组学和互补生化方法来表征SHS诱导的脑硝基蛋白质组，随后采用生物信息学/系统生物学方法分析来表征蛋白质相互作用图谱。将斯普拉格-道利大鼠暴露于SHS 5周，然后收集皮质组织。通过对脑裂解物蛋白质进行3-硝基酪氨酸（3-NT）免疫沉淀来进行硝基蛋白质富集。通过蛋白质印迹法验证蛋白质硝化，以确认硝基蛋白质的存在，并通过数据依赖型液相色谱-串联质谱（LC-MS/MS）进行无凝胶神经蛋白质组学分析作为补充。我们在3-NT富集样品中鉴定出29种差异表达蛋白；其中七种蛋白是SHS暴露所特有的。网络分析揭示了这些蛋白质与不同细胞过程的关联，包括氧化应激、ROS生成和细胞死亡相关途径。这证实了氧化应激机制与SHS的关联，这可能导致神经元损伤，而这一领域在吸烟方面尚未得到充分研究。
