AUF1在胞质DNA抑制细胞葡萄糖代谢中的作用研究

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稳定性参与胞质DNA引起的糖代谢应答反应。

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

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 enzymes. Real-time PCR also confirmed the above phenomena, suggesting that AUF1 may regulate glucose metabolism by stabilizing GLUT3 mRNA. (4) Both single-stranded and double-stranded ISD stimulation lead to a decrease in GLUT3 mRNA expression, suggested that GLUT3 might be a downstream effector in the regulation of glucose metabolism upon cytosolic DNA stimulation.Conclusions: AUF1 can bind to cytosolic DNA, and participate in the cytosolic DNA induced Glucose metabolic response, potentially through regulating the stability of GLUT3 mRNA.