丁烯醛对动脉内皮细胞氧化损伤的蛋白组学研究与生物信息学分析

Proteomics and bioinformatics analyses of oxidative damage of human arterial endothelial cells induced by crotonaldehyde

目的通过同位素标记相对与绝对定量蛋白质组学技术筛查丁烯醛导致人动脉内皮细胞损伤进程中的氧化损伤相关蛋白标记物。方法根据细胞活力损伤曲线确定导致90%细胞损伤所需的丁烯醛药物浓度(LD90)。利用蛋白质组学技术检测丁烯醛处理和未处理过的人动脉内皮细胞的蛋白质表达情况;采用生物信息学手段分析蛋白质数据;使用平行反应监测技术进行蛋白标记物的验证;通过检测线粒体膜电位与分光光度法测谷胱甘肽活性验证目标蛋白改变对氧化损伤功能的影响。结果以倍数变化>1.5的标准(上调>1.5倍或者下调至<67%)共鉴定出153个差异表达蛋白,其中氧化损伤相关差异表达蛋白质共12种:传感蛋白(A0A2P9AJA0)、激活信号辅整合素1复合体亚基2(B1AH59)、烟酰胺单核苷酸腺苷转移酶1(B1AN62)、cDNA FLJ58404(B4DRL9)、cDNA FLJ55250(B4DV58)、cDNA FLJ55007(B4DY35)、氧化酶(细胞色素1)装配蛋白样1(E7EVY0,OXA1L)、电子转移黄素蛋白α亚单位(H0YL12)、细胞色素C氧化酶亚基6A(O95101)、谷氨酸-半胱氨酸连接酶调节亚基(P48507)、细胞色素C氧化酶装配因子4(Q9NYJ1)、39S核糖体蛋白L3(P09001)。基因本体(GO)、京都基因和基因组百科全书(KEGG)富集分析显示差异蛋白明显富集的生物过程与碳水化合物、脂肪酸等有机物的代谢有关,而代谢过程则与ATP的产生、代谢氧化产物的产生和集聚、烟酰胺腺嘌呤二核苷酸(NAD)或烟酰胺腺嘌呤二核苷酸磷酸(NADP)的相互作用等密切相关。丁烯醛作用于人动脉内皮细胞后线粒体膜电位出现明显降低,谷胱甘肽活性出现了明显下降。蛋白网络互作结果显示,OXA1L、电子转移黄素蛋白亚单位α(ETFA)、线粒体核糖体蛋白L3(MRPL3)及谷氨酸-半胱氨酸连接酶修饰亚单位(GCLM)为4个关键的候选蛋白质。结论丁烯醛改变了人动脉内皮细胞的氧化损伤相关蛋白表达模式,表现为OXA1L、ETFA、GCLM蛋白表达升高,MRPL3表达下降。

Objective To study the protein markers related to oxidative damage involved in the human artery endothelial cells injury induced by crotonaldehyde using tandem mass tag(TMT)-based liquid chromatography-tandem mass spectrometry(LC-MS/MS)and bioinformatics analysis.Methods The concentration of crotonaldehyde required to cause 90%cell damage(LD90)was determined according to the cell viability damage curve.Proteomic analysis was used to detect the protein expression in crotonaldehyde-treated and untreated human arterial endothelial cells.The data on proteins which related with oxidative stress were analyzed by bioinformatics and confirmed by parallel reaction monitoring.The effect of target protein changes on oxidative function was confirmed by detecting mitochondrial membrane potential and measuring glutathione activity by spectrophotometry.Results A total of 153 significantly differentially expressed proteins were determined according to the criterion of multiple change>1.5,in which 12 were related to oxidative damage:sensor protein(A0A2P9AJA0),activating signal cointegrator 1 complex subunit 2(B1AH59),nicotinamide mononucleotide adenylyltransferase 1(B1AN62),cDNA FLJ58404(B4DRL9),cDNA FLJ55250(B4DV58),cDNA FLJ55007(B4DY35),oxidase(cytochrome C)assembly 1-like(OXA1L,E7EVY0),electron transfer flavoprotein subunit alpha(H0YL12),cytochrome C oxidase subunit VIa homolog(O95101),Glutamate-cysteine ligase regulatory subunit(P48507)and cytochrome C oxidase assembly factor 4 homolog(Q9NYJ1),39S ribosomal protein L3(P09001).The gene ontology(GO)analysis and Kyoto encyclopedia of genes and genomes(KEGG)analysis showed that the biological process of differentially expressed proteins was related to the metabolism of carbohydrates,fatty acids and other organic substances,while the metabolic process was closely related to the production of ATP,the production and aggregation of metabolic oxidation products,and the interaction of nicotinamide adenine dinucleotide(NAD)or nicotinamide adenine dinucleotide phosphate(NADP).Exposure to crotonaldehyde was found to be linked to reduced mitochondrial membrane potential and glutathione activity.Protein-protein interaction network analyses showed that OXA1L,electron transfer flavoprotein subunit alpha(ETFA),mitochondrial ribosomal protein L3(MRPL3)and glutamate-cysteine ligase modifier subunit(GCLM)were the four key protein biomarkers.Conclusion Crotonaldehyde significantly changes the protein expression pattern of human artery endothelial cells which associated with the oxidative stress,which is characterized by increased expression of OXA1L,ETFA,GCLM proteins and decreased expression of MRPL3.