2021 Volume 65 Issue 1 Pages 7-11
Cytosolic DNA derived from pathogen or host cells activates cyclic GMP-AMP synthase (cGAS) to produce cyclic GMP-AMP dinucleotide (cGAMP). The binding of cGAMP to a downstream adaptor protein, stimulator of interferon genes (STING), induces innate immune response. Here, we reported two useful methods to analyze the cGAS-cGAMP-STING signaling pathway. One is a sensitive in vitro assay to detect cGAMP using clear-native PAGE, based on its ability to induce STING dimerization. Using this method, we successfully detected cGAMP in the fetal liver and spleen of DNase II deficient mice, which cause a strong inflammatory response that leads to lethal anemia or polyarthritis. Another is an improved proximity-dependent biotin identification (BioID) method, which is a powerful technology to identify protein-protein interactions in living cells. By using the improved BioID method, we identified previously known and unknown interactors of STING, such as several palmitoyl transferases, interferon regulatory factor 5 (IRF5) and interferon-induced transmembrane protein 3 (IFITM3). The STING interactome obtained in this method will provide new insights into the cGAS-cGAMP-STING signaling network.