几个能量代谢相关蛋白在HeLa细胞自噬过程中表达下调  

Several Catabolic Proteins are Down-regulated in Starvation Induced Autophagy in HeLa Cells

作　　者：刘丹慧[1] 韩姗姗[1] 李潞云[1] 曲业敏[1] 林虹[1] 文杰[1] 吕建新[1] 

机构地区：[1]温州医学院细胞与分子医学研究所浙江省医学遗传学重点实验室蛋白质组学研究所,温州325035

出　　处：《中国细胞生物学学报》2011年第6期651-657,共7页Chinese Journal of Cell Biology

基　　金：浙江省自然科学基金(No.Y2090378);浙江省教育厅项目(No.20070909);引进大院名校共建创新载体以奖代补基金(No.2008E80046);温州医学院校内课题(No.QTJ06011)资助项目~~

摘　　要：自噬是真核细胞中的一种保守的代谢信号通路。人们已经知道自噬与肿瘤发生等疾病密切相关,但对于自噬的分子机制仍然不是很清楚。鉴定更多的自噬相关蛋白对于进一步阐明自噬的分子机制具有重要意义。该研究使用饥饿法处理HeLa细胞,通过电镜观察以及检测自噬标记蛋白LC3-I的转换,证实HeLa细胞发生了明显的自噬。之后,使用双向电泳结合串联质谱分析鉴定细胞自噬时发生变化的蛋白质。结果发现果糖二磷酸醛缩酶A、GAPDH和ATP合成酶O亚基的量在HeLa细胞发生自噬后明显降低。实时定量PCR结果证明饥饿诱导后,这三种蛋白的mRNA水平都发生了明显的下降。使用自噬抑制剂3-Methyladenine预处理HeLa细胞后再行饥饿,三种蛋白mRNA的表达水平与正常细胞相当而明显高于饥饿诱导的细胞。结果表明这三种蛋白在饥饿诱导的自噬中表达下调,其分子机制还有待进一步研究。Autophagy is a conversed catabolic pathway that plays an important role in maintaining cell homeostasis by degrading damaged cytosolic components and redundant proteins in eukaryotic cells. Autophagy plays a protective role against various human diseases, including cancer. Although many autophagy related proteins have been investigated, there are many unsolved problems about the mechanism of the occurrence and regulation of autophagy. In this study, autophagy was induced in HeLa cells by starvation. The occurrence of autophage was confirmed with transmission electron microscopy and LC3-1 conversion, which are hallmarks of autophagy. We used 2-DE coupled with LC-MS/MS to analyze the differential protein expression in starved HeLa cells. We found fructose-bisphosphate aldolase A, GAPDH and ATP synthase O subunit were significantly decreased in starvation-induced autophagy. This was confirmed with real-time quantitative PCR. To address if these proteins were directly related with autophagy, HeLa cells were pretreated with the autophagy inhibitor 3-MA, and the total RNA were extracted for real-time quantitative PCR after starvation. The data revealed 3-MA can rescue the mRNA decrease of fructose-bisphosphate aldolase A, GAPDH and ATP synthase O subunit. Taken together, our results indicate that these proteins may be involved in the regulation of autophagy, and the mechanism need further investigation.

关 键 词：自噬 饥饿 双向电泳 质谱 差异蛋白 

分 类 号：R346[医药卫生—基础医学]