ISGylation of zebrafish STING at lysines 221 and 276 activates innate immunity.

The interferon gene stimulator (STING) is an endoplasmic reticulum (ER)-resident protein that plays a crucial role in the immune response to microbial infections. ISGylation, a form of post-translational modification, regulates innate immunity in mammals. However, the regulatory mechanism of ISGylation on STING in fish remains largely unknown. In this paper, we identified that ISG15-assembled ISGylation is critical for the STING-mediated innate immune response in zebrafish. Upon screening lysine sites, we discovered that ISGylation of STING was catalyzed at lysine residues 221 and 276. When these lysines were mutated to arginine (forming STING^K221R or STING^K276R), the oligomerization of STING and the STING-mediated innate immune response were diminished. This evidence suggests that by reducing the phosphorylation of STING, interferon regulatory factor 3 (IRF3), and TANK-binding kinase 1 (TBK1), there is ultimately a decrease in the induction of type I interferon (IFN I). Moreover, the inhibition of STING ISGylation individually promoted its K48-linked ubiquitination, decreased its K63-linked ubiquitination, and phosphorylation. These results suggest that ISGylation can protect STING from ubiquitination and subsequent degradation. STING is primarily colocalized with the Golgi apparatus and endoplasmic reticulum, ensuring its normal function. However, mutations in STING, such as STING^K221R or STING^K276R, impair its accurate localization and oligomerization. This study, to our knowledge, provides novel insights into the role of ISGylation in STING-mediated innate immune responses in fish.