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The Role of AUF1 in Cytosolic DNA Induced Cellular Glucose Metabolic Response |
XIE Lin-na1(),BU Jing-jing2,ZHENG Min2 |
1 Fujian Vocational College of Bioengineering, Fuzhou 350003,China 2 Fujian Medical University, Fuzhou 350108,China |
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Abstract 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.
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Received: 28 April 2019
Published: 17 December 2019
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Corresponding Authors:
Lin-na XIE
E-mail: bunnylinda@qq.com
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