A quantitative method to monitor STING degradation with dual-luciferase reporters

• Published in: Cell Structure and Function Vol. 50; no. 1; pp. 115 - 124
• Main Authors: Shindo, Ruri, Mukai, Kojiro, Taguchi, Tomohiko, Hongo, Kazune, Shoji, Tsumugi, Shinojima, Ayumi, Koide, Shogo, Kuchitsu, Yoshihiko, Sato, Kanako
• Format: Journal Article
• Language: English
• Published: Japan Japan Society for Cell Biology 01.01.2025; Japan Science and Technology Agency
• Subjects: Degradation; Flow cytometry; Genes, Reporter; Golgi apparatus; HEK293 Cells; Humans; Inflammation; Inflammatory diseases; innate immunity; Interferon; Localization; luciferase; Luciferases - genetics; Luciferases - metabolism; lysosomal degradation; Lysosomes; Lysosomes - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; membrane traffic; Microscopy; Plasmids; Proteins; Proteolysis; STING; Western blotting; luciferase; STING; innate immunity; lysosomal degradation; membrane traffic
• ISSN: 0386-7196; 1347-3700; 1347-3700
• DOI: 10.1247/csf.25011

Stimulator of interferon genes (STING) triggers the type I interferon and inflammatory responses against a variety of DNA pathogens, which is essential to limiting viral infection and replication. STING activates the downstream kinase TBK1 at the trans-Golgi network (TGN) and is degraded at lysosomes through a process called lysosomal microautophagy. Impaired STING targeting to lysosomes results in the prolonged inflammatory signal, which may be associated with a variety of neurodegenerative and autoinflammatory diseases. Thus, development of methods to quantify STING degradation helps understand the mechanism of lysosomal microautophagy and its related diseases. Here we report a quantitative method to monitor STING degradation with two luciferases, firefly luciferase (FLuc) and Nanoluciferase (NLuc). The expression plasmid is composed of FLuc, a P2A self-cleavage site, and NLuc-tagged STING. FLuc intensity reflects the total amount of translated protein, serving as an internal control, while NLuc intensity corresponds to the amount of STING. Comparison of the NLuc/FLuc ratios at different time points after STING stimulation revealed the kinetics of decay of STING levels in live cells. This method should provide a useful complement to western blotting and fluorescence-activated cell sorter (FACS) analysis presently used to monitor STING degradation.Key words: innate immunity, STING, membrane traffic, lysosomal degradation, luciferase