Fig. 2: ISGylation at four key lysine residues (K236, K338, K347, and K370) promotes STING stability through the USP18-HERC5 axis.

A Detection of STING ISGylation and screening of E3 ligases. Flag-STING was co-transfected with UBE1L-Myc (E1), UBCH8-Myc (E2), Myc-ISG15, and either ARIH1-Myc-His, EFP-Myc-His, or HERC5-Myc-His into HEK-293T cells. Cells were harvested and lysed 24 h post-transfection, and then lysates were subjected to immunoprecipitation using Flag-beads. Western blot analysis with an ISG15 antibody was performed to detect ISG15-modified STING bands. B De-ISGylation of STING by USP18. Flag-STING along with UBE1L-Myc-His (E1), UBCH8-Myc-His (E2), HERC5-Myc-His (E3), and Myc-ISG15 were co-transfected with GFP-tagged USP18-WT or the catalytically inactive USP18-C64S mutant into HEK-293T cells. Twenty-four hours post-transfection, cells were subjected to an ISGylation assay, performed as described above. C Verification of STING ISGylation using an ISGylation-deficient mutant ISG15-C2. Flag-STING along with E1, E2, and E3 plasmids as in (B) were co-transfected with either Myc-ISG15-WT or Myc-ISG15-C2 into HEK-293T cells. ISGylation experiments were performed as described in B. D ISGylation of endogenous STING was analyzed by immunoprecipitation with STING antibody followed by western blot with anti-ISG15 antibody and STING antibody in H1299 cells in the presence of diABZi (10 µM for 4 h). E The effect of ISG15-C2 on STING protein stability. H1299 cells were transfected with plasmids expressing ISG15-WT or ISG15-C2. After 24 h, cells were treated with diABZi (1 μM for 12 h). ISGylation experiments were performed as described in B. F The impact of the ISG15-C2 on IFN-β transcription. Transfection was performed as described above and changes in IFN-β mRNA were measured by RT-qPCR. G Screening for ISGylation sites on STING. Flag-STING WT or STING-K only mutant plasmid was co-transfected with E1, E2, E3, and Myc-ISG15. ISGylation experiments were performed as described in B. H The effect of specific lysine retention on STING degradation. H1299 cells were transfected with plasmids expressing single retention of nine lysine (K-only) residues of STING, along with Myc-ISG15. Twenty-four hours post-transfection, cells were treated with diABZi (1 μM for 12 h), and western blot was used to assess the degradation differences of STING protein. I The effect of STING-4KR mutation on STING and p-IRF3 protein stability. H1299 cells were transfected with either STING-WT or STING-4KR plasmid. Twenty-four hours post-transfection, cells were treated with diABZi (1 μM) at various time points (3, 6, and 12 h). Western blot analysis was conducted to detect differences in STING protein degradation, followed by densitometric analysis of the bands. J Schematic representation of the regulation of STING protein stability by ISGylation. ISGylation at K236, K338, K347, and K370 are essential for STING protein stability at rest and post-diABZi treatment. The data presented is from a representative experiment, selected from three independent experiments. Data were presented as mean ± SEM, comparisons were conducted using two-way ANOVA for multiple comparisons, with **** indicating p < 0.0001 and n.s indicating not significant.