活化的大鼠肝星状细胞赖氨酸乙酰化蛋白质组学分析

Proteomics analysis on lysine acetylation of proteins in actived hepatic stellate cells of rats

目的对活化的大鼠肝星状细胞(hepatic stellate cells,HSCs)进行赖氨酸乙酰化(lysine acetylation,Kac)蛋白质组学分析。方法于清洁级雄性SD大鼠中提取并培养原代HSCs,分为对照组(培养<2天)和活化组(培养≥10天)。对两组细胞进行裂解,iTRAQ/TMT标记后的成对肽段样品采用乙酰化抗体进行亲和富集,应用高分辨率LC-MS/MS技术进行质谱鉴定。最后对质谱鉴定的乙酰化蛋白质进行生物信息学分析。结果应用LC-MS/MS技术、GO注释和KEGG通路等生物信息学分析方法,共确定309种蛋白质中的470个Kac位点。对其中150个蛋白质的231个Kac位点进行肽段定量研究,与静息的HSCs相比,在活化的HSCs中有109个蛋白质的乙酰化肽段数目增加,18个蛋白质的乙酰化肽段数目减少,并涉及多种GO分类和KEGG通路。乙酰化蛋白质广泛定位于胞质(38.1%)和线粒体(30.2%)。基于GO富集的聚类分析显示Kac位点主要与能量代谢相关;基于KEGG通路的聚类分析显示这些蛋白质广泛参与三羧酸循环,与线粒体关系密切。结论首次对HSCs的Kac蛋白质进行质谱分析,并对发生乙酰化的蛋白质位点进行量化鉴定,为揭示蛋白质乙酰化在HSCs活化过程中的作用提供了一定的基础,并为抑制肝纤维化提供了潜在的分子靶标。

Objective To investigate the lysine acetylation（Kac）of proteins in activated hepatic stellate cells（HSCs）of rats. Methods Pathogen-free male SD rats were used for the isolation of HSCs,and the primary cells were divided into control group（〈2 days of culture）and active group（≥10 days of culture）.In the two groups,iTRAQ/TMT labeling were performed on the samples,and then the labelled proteins were followed by immune-affinity isolation of acetyl-peptides through anti-acetyl lysine antibodies.2D nano-LC-MS/MS was used to identify acetyl-proteome of HSCs.Finally,the quantitative analysis of acetylation sites and bioinformatics analysis were carried out. Results A total of 309 acetylated proteins with 470 Kac sites were identified in HSCs,of which 150 proteins with 231 Kac sites were quantified.Compared with quiescent HSCs,acetyl-peptides of 109 proteins showed significant increase in activated HSCs,and acetyl-peptides of 18 proteins showed significant reduce in activated HSCs.Bioinformatics and physiological analyses of detected proteins suggested that these acetylated proteins were widely located in cytosol and mitochondria,which contain 38.1% and30%,respectively.GO enrichment-based clustering analysis indicated that Kac of these proteins may strictly regulate energy metabolism.In addition,KEGG pathway enrichment-based clustering analysis suggested that these protein significantly up-regulated in TCA cycle,which have intense relationship with mitochondria. Conclusions Kac of proteins in HSCs were investigated for the first time,and quantitative comparison of acetyl-proteome before and after HSCs activation were extensively studied in order to reveal the relationship between acetylation and HSCs activation,and to provide the potential target spots to inhibit the fibrosis of liver.