AUF1在胞质DNA抑制细胞葡萄糖代谢中的作用研究  

The Role of AUF1 in Cytosolic DNA Induced Cellular Glucose Metabolic Response

作　　者：谢琳娜 卜静静 郑敏[2] XIE Lin-na;BU Jing-jing;ZHENG Min(Fujian Vocational College of Bioengineering,Fuzhou 350003,China;Fujian Medical University,Fuzhou 350108,China)

机构地区：[1]福建生物工程职业技术学院,福州350003 [2]福建医科大学,福州350108

出　　处：《中国生物工程杂志》2019年第11期54-61,共8页China Biotechnology

摘　　要：目的:探讨AUF1在胞质DNA引起的细胞葡萄糖代谢应答中的作用及其机制。方法:(1)用核质分离技术分离细胞核与细胞质,并通过生物素-亲和素亲和层析技术分离细胞质中与胞质DNA(ISD)结合的蛋白质,然后通过"银染-质谱"和"复合物-质谱"技术鉴定出差异蛋白——AUF1。再利用体外结合实验验证AUF1与胞质DNA的相互作用。(2)在胞质DNA刺激后,通过ATP检测试剂盒和CCK8细胞氧还活力检测试剂,比较野生型细胞和基于CRISPR/Cas9技术的AUF1基因敲除细胞中葡萄糖代谢应答情况。(3)通过半定量PCR技术,在野生型、基因敲除AUF1、基因敲除后回补AUF1或空载体的四类细胞中检测葡萄糖转运蛋白GLUTs以及葡萄糖代谢相关酶的mRNA表达情况,筛选出与细胞糖代谢相关的AUF1下游效应分子——GLUT3。进而用实时荧光定量PCR进行验证。(4)通过半定量和荧光定量PCR分析胞质DNA刺激下GLUT3的mRNA变化情况,分析胞质DNA的刺激是否影响GLUT3的mRNA表达。结果:(1)两次质谱分析均发现AUF1能与ISD结合。体外结合实验也证实,不论是原核表达的GST-AUF1还是真核细胞表达的GFP-AUF1均能与单链和双链的ISD相结合。(2)基因敲除AUF1后的HEK293细胞在用胞质DNA刺激后,胞内的ATP水平和对CCK8的还原能力都明显高于野生型细胞。提示AUF1基因敲除细胞内的葡萄糖代谢不受胞质DNA刺激所抑制,说明AUF1很可能参与了胞质DNA对细胞糖代谢的调节。(3)半定量PCR技术检测发现在AUF1敲除的细胞中GLUT3的mRNA明显减少,而其他的GLUT家族成员和代谢酶则没有显著差异。实时荧光定量PCR证实上述现象,提示AUF1很可能通过稳定GLUT3的mRNA参与葡萄糖代谢的调节。(4)无论是单链还是双链ISD刺激后的细胞中,GLUT3的mRNA均减少,说明GLUT3可能是胞质DNA对糖代谢的调节过程中的一个下游效应分子。结论:AUF1能与胞质DNA结合,很可能通过调节下游GLUT3的mRNA稳定性�Objective:The molecular mechanism of cytosolic DNA induced cellular glucose metabolic response was aimed to unravel.Methods:(1)The nucleus and cytoplasm were separated by fractionation,and the protein bound to cytosolic DNA(ISD)was isolated using biotin-avidin affinity chromatography.The differentially expressed protein,AUF1 was identified by silver staining followed by mass spectrometry analysis or directly by complex-mass spectrometry analysis.The interaction between AUF1 and ISD was verified by pull-down assay.(2)ATP assay and CCK8 analysis were performed to evaluate the cytosolic DNA induced cellular glucose metabolic response in wildtype and AUF1 knockout cells,which was generated by CRISPR/Cas9 technology.(3)The mRNA expression of glucose transporters GLUTs and key enzymes in the process of glucose metabolism were detected by semi-quantitative PCR in four types of cells:wild type HEK293 cells,AUF1 knockout HEK293 cells,and AUF1 knockout cells reconstituted with AUF1 or empty vector as controls.GLUT3 was identified as one of the downstream effectors of AUF1.Real-time PCR was also performed to verify the results.(4)GLUT3 mRNA under the stimulation of cytosolic DNA was analyzed by semi-quantitative and real-time PCR.Results:(1)Both mass-spectrometry analyses showed that AUF1 could bind to ISD.In vitro binding assays also confirmed that both GST-AUF1 expressed in prokaryotic cells and GFP-AUF1 expressed in eukaryotic cells could bind to single-stranded and double-stranded ISDs.(2)Upon cytosolic DNA stimulation,intracellular ATP levels and reductive capabilities of AUF1-/-HEK293 cells were higher than wild-type cells.It suggested that the glucose metabolism in AUF1 knockout cells is not inhibited by cytosolic DNA stimulation,and AUF1 may be involved in cytosolic DNA induced cellular glucose metabolic response.(3)Semi-quantitative PCR analysis showed that GLUT3 mRNA expression was significantly reduced in AUF1 knockout cells,while there were no significant differences among other GLUT family members and metabolic

关 键 词：AUF1 胞质DNA 葡萄糖代谢 GLUT3 

分 类 号：R34[医药卫生—基础医学]