Identification and Analysis of Interacting Proteins Regulating IFNβ Expression in PPARγ Activated and Inhibited States
MA Zhuo-Yuan1,2, YANG Bin1,2, CHEN Yun-Long1,2, FAN Gang1,2, WU Xiao-Long1,2, WANG Xi-Yin1,2, HUA Jin-Lian1,2, WANG Yan1,2, ZHANG Yong-Qiang3,*, ZHANG Shi-Qiang1,2,*
1 College of Veterinary Medicine/Shaanxi Stem Cell Engineering Research Center, Northwest A&F University, Yangling 712100, China; 2 Key Laboratory of Livestock Biology, Northwest A&F University, Yangling 712100, China; 3 China Animal Health and Epidemiology Center, Qingdao 266032, China
Abstract：Peroxisome proliferator activated receptor γ (PPARγ) is specifically overexpressed in alveolar macrophages and has metabolic and immunoregulatory roles. It is not clear which interacting proteins of PPARγ are involved in the regulation of typeⅠinterferon production induced by innate immunity. In this study, wild boar (Sus scrofa) lung cell line (WSL) with stable overexpression of PPARγ-3×Flag was constructed using lentiviral vector system. Taking this cell strain as a model, fluorescence quantitative PCR assay revealed that the addition of the PPARγ agonist rosiglitazone significantly reduced the level of interferon β (IFNβ) expression induced by stimulating with the dsDNA mimic Poly (dA:dT); In contrast, addition of the PPARγ inhibitor T0070907 significantly increased the level of IFNβ expression induced by Poly (dA:dT) stimulation. Further, the PPARγ-3×Flag protein complex regulating the above process was precipitated by immunomagnetic beads of anti-FLAG protein. The PPARγ-3×Flag protein complexes enriched in cytoplasmic proteins and nuclear proteins were digested into peptides and analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS), respectively. The results showed that there were 101 cytoplasmic proteins and 95 nucleoproteins interacting specifically with PPARγ when the PPARγ agonist rosiglitazone was added. Under the condition of adding PPARγ inhibitor T0070907, there were 24 cytoplasmic proteins and 14 nuclear proteins that specifically interacted with PPARγ. GO and KEGG analysis showed that specific PPARγ interactions of cytoplasmic proteins and nuclear proteins were related to translation regulation. This study provides a new clue to further study the mechanism of regulating of IFNβ expression by PPARγ.
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