THE EFFECTS OF ENDOPLASMIC RETICULUM-GOLGI VESICULAR TRAFFIC ON STING ACTIVATION

Project no. 23761 · Status: Kapanmış (Yayın bekleniyor) · 2022

Summary

Recognition of DNA in the cytoplasm as an immunostimulatory molecule is an evolutionarily conserved mechanism to initiate the innate immune response against pathogens. Cytosolic DNA is detected by cyclic GMP-AMP synthase (cGAS) and the second messenger cyclic GMP-AMP (cGAMP) is synthesized. After cGAMP binding, STING in the endoplasmic reticulum membrane is activated and leaves the endoplasmic reticulum to the Golgi. It activates TANK-binding kinase 1 (TBK1) in the Golgi and phosphorylates the interferon regulatory transcription factor IRF3. Phosphorylated IRF3 dimerizes and translocates to the nucleus, where it induces the expression of inflammatory genes, including type-I interferon (IFN) and IFN-stimulated genes. This thesis aims to elucidate the role of endoplasmic reticulum-golgi vesicular traffic in the STING activation.In this study, whole transcriptome analysis was performed after activation of the cGAS-STING pathway in Calu-1 cells. As a result of transcriptome analysis, RNF149, whose expression increased significantly, was determined as a candidate gene and its role in STING activation was investigated by knocking it out using the CRISPR/Cas9 method. The expression of genes involved in the cGAS-STING pathway was examined by western blot, and the expression of interferon and interferon-stimulated genes was examined by qRT-PCR analysis.As a result of western blot analysis, an increase in STING, TBK1, and IRF3 expression and a decrease in p-STING, p-TBK1, and p-IRF3 expression were observed in RNF149 knock-out (RNF149-KO) cells treated with agonist for 30 minutes compared to wild-type Calu-1 (WT) cells. When agonist was applied for 1 hour, no significant change was observed in the expressions of STING, p-TBK1, p-IRF3; however an increase in the expressions of p-STING, TBK1 and IRF3 was determined. When agonist was applied for 4 hours, an increase was observed in the expressions of p-STING, p-TBK1, TBK1, IRF3; but no change was observed in the expressions of STING and p-IRF3. It was determined that there was an increase in the expression of cis-Golgi matrix protein GM130 in RNF149-KO cells. When RNF149-KO and wild type Calu-1 cells were compared according to the qRT-PCR results, a significant decrease in IFI44 and IL6 mRNA expressions was determined after 1 hour of agonist application whereas there was no significant change in IFNB1, IFIT2 and ISG15 expressions. After 4 hours agonist application, there was a significant decrease in IFNB1 and IL6 mRNA expressions, while there was no significant change in IFIT2, IFI44 and ISG15 expressions. After 8 hours of agonist application, there was a significant increase in IFIT2 mRNA expression, while there was no significant change in IFNB1, IFI44, ISG15 and IL6 expressions.

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