基于iTRAQ的蛋白质组学技术对Drp1基因过表达挽救帕金森病模型果蝇的机制研究

Rescue effect of Drpl gene over-expression on drosophila models of Parkinson＇s disease： a mechanism research by iTRAQ-based proteomic analysis

目的应用iTRAQ技术探讨线粒体动力相关蛋白1（Drp1）基因过表达挽救PD模型果蝇的蛋白质组学变化以及挽救效应的分子机制。方法将各果蝇品系分别杂交构建出不同实验果蝇：正常组、PD模型组（PINK1B9转基因疾病果蝇）和PD挽救组（Drp1基因过表达PD模型果蝇），并观察果蝇翅膀形态、运动能力及计算异翅率、飞行率。提取果蝇蛋白组分，以iTRAQ技术联用串联质谱分析蛋白质组的整体变化情况，并将各组果蝇蛋白鉴定结果分别两两比较获得差异蛋白，最后将鉴定得到的差异蛋白进行GO功能富集分析和KEGG信号通路富集分析。结果PD挽救组的异翅率与PD模型组相比明显降低（2.60%±0.47% vs. 82.40%±12.47%），飞行率与PD模型组相比明显提高（89.70%±7.76% vs. 3.30%±1.69%），差异均有统计学意义（P〈0.05）。蛋白质组学分析结果共鉴定得到3630个蛋白，其中PD模型组与正常组间鉴定得到的差异蛋白共282个，主要是铁离子相关蛋白；PD挽救组与PD模型组间鉴定得到的差异蛋白共170个，主要是锌离子相关蛋白。共同差异蛋白的信息显示Drp1基因过表达后逆转了绝大多数PD模型中相对正常果蝇异常表达的蛋白，其中21%的蛋白具有金属离子结合功能，且主要是锌离子结合功能。结论金属离子代谢可能在PD发病及Drp1基因过表达挽救PD过程中有重要作用，尤其是锌离子代谢可能在挽救效应中起主要作用。

ObjectiveTo investigate the rescue effect of Drp1 gene over-expression on drosophila models of Parkinson＇s disease （PD） and their specific mechanism using iTRAQ-based proteomic technology.MethodsThe drosophilae from 3 groups, control group, PD group （PINK1 mutant）, and rescue group （PINK1 mutant＋Drp1 over-expression） were cultured at routine ways. The wing shape and movement ability of drosophilae were observed, and the percentages of drosophilae having abnormal wings and normal flight were calculated. The proteomic changes were determined by iTRAQ technology; the functions and signaling pathways of the differential expressed proteins were analyzed by GO and KEGG pathway enrichment analyses.ResultsPercentage of drosophilae having abnormal wings in the rescue group （2.60%±0.47%） was significantly decreased as compared with that in the PD group （82.40%±12.47%, P〈0.05）, and the percentage of drosophilae having normal flight in the rescue group （89.70%±7.76%） was significantly increased as compared with that in the PD group （3.30%±1.69%, P〈0.05）. A total of 3630 proteins were identified using iTRAQ; 282 differential expressed proteins between the PD group and control group were detected, which mainly were iron ion related proteins; 170 differential expressed proteins between the PD group and rescue group were detected, which mainly were zinc ion related proteins. GO and KEGG pathway enrichment analyses on these co-differential expressed proteins revealed that 21% proteins had metal ion binding activity, especially zinc ion.ConclusionAbnormal iron homeostasis, especially zinc homeostasis, participates in PD pathophysiological processes and over-expressing Drp1 rescued PD processes.