甘酰胺tRNA合成酶对奶牛乳腺乳蛋白合成的调控机理  

Regulation of GlyRS on Milk Protein Synthesis in Dairy Cow Mammary Epithelial Cells and Its Mechanism

作　　者：骆超超[1] 王春梅[1] 李庆章[1] 高学军[1] 

机构地区：[1]东北农业大学

出　　处：《科技创新导报》2016年第8期168-169,共2页Science and Technology Innovation Herald

摘　　要：乳蛋白合成机理是重要的生命科学基础问题之一,目前已发现催乳素通过JAK/STAT5信号通路在转录水平调控乳蛋白合成,氨基酸通过m TOR/S6K1信号通路在翻译水平调控乳蛋白合成。但乳腺细胞内乳蛋白合成还有哪些重要的信号分子参与转录和翻译调控,什么分子是氨基酸等营养素的"分子传感器",细胞核和细胞液之间的串话如何应答氨基酸信号从而调节乳蛋白的合成等,仍是尚待解决的重要科学问题。该实验较系统的研究了甘氨酰胺合成酶作为一个新的信号分子,接受蛋氨酸信号,从转录水平和翻译水平对奶牛乳腺上皮细胞中β-酪蛋白(CSN2)合成的调节作用及机理。实验既为人们弄清楚氨基酸对乳蛋白的调节及作用机理提供基本理论依据,也为人们全面了解乳蛋白合成的网络信号通路提供新的视野。该研究采用组织块培养法培养并纯化体外培养的奶牛乳腺上皮细胞,用蛋白质免疫印记和免疫荧光检测细胞中角蛋白18(CK18)和CSN2的表达以鉴定细胞纯度和泌乳功能。实验通过在培养液中添加0.6 mmol/L的蛋氨酸,建立细胞泌乳模型用实时荧光定量PCR、WB和IF等方法,检测添加蛋氨酸泌乳模型中,Gly RS的表达与定位情况。结果表明蛋氨酸在促进细胞泌乳时,Gly RS表达显著上升(<0.01),并且Gly RS入核也显著增加(<0.01)。这说明,在蛋氨酸上调CSN2表达过程中,Gly RS可能是一个重要的调节因子。对Gly RS进行过表达与干扰,利用q RT-PCR、WB和IF等方法检测m TOR、p-m TOR、Stat5a、p-Stat5a、S6K1、p-S6K1、4EBP1、p-4EBP1和CSN2的表达,用CASY细胞活力分析仪和流式细胞仪分析测定细胞活力、细胞数和细胞的增殖率。结果显示,Gly RS过表达和干扰后,检测的磷酸化蛋白和CSN2均分别显著上升和下降(<0.01),细胞活力、细胞数和细胞的增殖率也分别显著增加和减少(<0.01),而非磷酸化蛋白无显著变化(>0.05);添加蛋氨�The mechanism of milk protein synthesis is one of the important foundation problems of life science. In recent years, obvious progress of this problem has been made by some research. At present, we found that the transcription process of milk protein synthesis was regulated by prolactin through the JAK/STAT5 signaling pathways and the translation process of it was regulated by amino acids through the mT OR/S6K1 signaling pathways. But, which other important signaling molecules involved in transcription and translation of milk protein synthesis in mammary gland cells？ What molecule are the ＂molecular sensors＂ of amino acids and other nutrients and how the nucleus and cytoplasm reply the amino acid signal are still the important scientific problems that needed to be solved in lactation biology nowadays. In this study, a comprehensive study of glycyl-tR NA synthetase（GlyR S） regulation the synthesis of β-casein（CSN2） as a new signal molecule reply the methionine signal in dairy cow mammary epithelial cells and its mechanism were completed. Both the basic theory of milk protein synthesis regulated by amino acids and the new horizon of understanding the signaling pathway network of milk protein synthesis were provided by this study. Comprehensive these experimental results, GlyR S was an important signal molecule in the process of methionine regulated the lactation in dairy cow mammary gland epithelial cells. The mechanism of regulation by GlyR S was as follows：The T544 and S704 of GlyR S were phosphorylated after GlyR S accept the methionine signal in the cytoplasm,then a part of GlyR S combined with eI F2 D in the cytoplasm and raised the expression of CSN2 at translation level, the other part of GlyR S were guided into the nucleus by NLS, and these GlyR S were spliced at the same time. Then, the C-terminal parts of the spliced GlyR S combined with NFκB1 in the nucleus and promote the combination of NFκB1 with CSN2 promoter, and finally raised the expression of CSN2 at transcription level.

关 键 词：甘氨酰胺合成酶 泌乳信号通路 Β-酪蛋白 奶牛乳腺上皮细胞 蛋氨酸 

分 类 号：Q522.02[生物学—生物化学]