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

doi:10.13523/j.cb.20191106

China Biotechnology

2019, Vol. 39

Issue (11): 54-61 DOI: 10.13523/j.cb.20191106

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.

Key words： AUF1 Cytosolic DNA Glucose metabolic response GLUT3

Received: 28 April 2019 Published: 17 December 2019

ZTFLH:

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Corresponding Authors: Lin-na XIE E-mail: bunnylinda@qq.com

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Cite this article:

XIE Lin-na,BU Jing-jing,ZHENG Min. The Role of AUF1 in Cytosolic DNA Induced Cellular Glucose Metabolic Response. China Biotechnology, 2019, 39(11): 54-61.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20191106 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I11/54

Fig.1 Mass spectrometry and in vitro pull-down assay identified AUF1 as a single/double stranded ISD binding protein (a) Fractionation of nucleus and cytosol (b) Cytosolic DNA binding proteins were identified using biotin-avidin affinity chromatography and SDS-PAGE followed by silver staining and mass spectrum. AUF1 was found in #1 band (c) Pull-down assay verified that ISD interacts with AUF1 expressed in either prokaryotic or eukaryotic cells Ds:Double stranded; ss:Single stranded

Fig.2 AUF1 is involved in cytosolic DNA induced cellular energy response (a) AUF1 knockout HEK293 cells were generated by CRISPR/Cas9 technology, and verified by Western blot (b) Intracellular ATP levels were examined in cells stimulated as indicated (c) CCK8 assays were performed in cells treated as indicated

Fig.3 GLUT3 is regulated by AUF1 (a) Reconstitution of AUF1 in AUF1 knockout HEK293 cells (b) After RNA extraction and reverse transcription, primers were designed using full-length gene sequences as templates, and the expression of each gene was analyzed by semi-quantitative PCR (c) Using SybrGreen as a probe, the expression of GLUT3 was detected by real-time PCR

Fig.4 Cytosolic DNA stimulation decreases GLUT3 mRNA expression (a) HEK293 cells were stimulated as indicated, followed by RNA extraction and reverse transcription. Primers were designed using full-length GLUT3 gene sequence as a template, and the GLUT3 mRNA expression levels were analyzed by semi-quantitative PCR (b) Using SybrGreen as a probe, the expression of GLUT3 was examined using real-time PCR


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