Fig 2: TRIM18 recruits PPM1A to dephosphorylate TBK1 for its inactivation and black the interactions of TBK1 with its upstream adaptors. a Immunoblot (IB) analysis of total and phosphorylated (p-) IRF3 as well as ß-actin in lysates of WT and TRIM18 KO BMDM infected for 1 h without (Mock) or with CVB3 or adenovirus at an MOI of 5. b Immunoblot analysis of endogenous proteins TRIM18 and PPM1A precipitated with anti-PPM1A, or immunoglobulin G (IgG) from whole-cell lysates of WT and TRIM18 KO BMDM. c Schematic diagram showing full-length PPM1A (Full) and serial truncations of PPM1A with deletion of various domain (left margin); numbers at ends indicate amino acid positions (top). M1, the catalytic domain; M2, the C-terminal no catalytic domain. d Immunoblot analysis of purified HA-tagged full-length PPM1A and serial truncations of PPM1A with deletion of various domains alone with anti-HA antibody (top blot) or after incubation with Myc-tagged TRIM18 and immunoprecipitation with anti-Myc antibody (second blot), and immunoblot analysis of purified Myc-tagged TRIM18 with anti-Myc antibody (third blot) or after incubation with Myc-tagged TRIM18 and immunoprecipitation with anti-Myc antibody (bottom blot). e Schematic diagram showing full-length TRIM18 (Full) and serial truncations of TRIM18 with deletion (?) of various domain (left margin); numbers at ends indicate amino acid positions (top). RING, the really interesting new gene domain; BBOX, the B-box zinc-finger domain; BBC, the B-box C-terminal domain; FN3, the fibronectin type 3 domain; SPRY, the Sp1A kinase and Ryanodine receptors domain. f Immunoblot analysis of purified HA-tagged full-length TRIM18 and serial truncations of TRIM18 with deletion of various domains alone with anti-HA antibody (top blot) or after incubation with Myc-tagged PPM1A and immunoprecipitation with anti-Myc antibody (second blot), and immunoblot analysis of purified Myc-tagged PPM1A with anti-Myc antibody (third blot) or after incubation with Myc-tagged PPM1A and immunoprecipitation with anti-Myc antibody (bottom blot). g Immunoblot analysis of total and phosphorylated (p-) TBK1, PPM1A, TRIM18 as well as ß-actin in lysates of WT and TRIM18 KO BMDM infected for 1 h without (Mock) or with CVB3 or adenovirus at an MOI of 5. h Immunoblot analysis of endogenous proteins STING, MAVS, TBK1 and TRIM18 from whole-cell lysates (Input) or precipitated with anti-TBK1 antibody (IP: TBK1) from lysates of WT and TRIM18 KO BMDM left infected (Mock) or infected with CVB3 or adenovirus at MOI of 10 for 6 h. The position of protein markers (shown in kDa) is indicated on the right. Data are representative of three independent experiments

Fig 3: Schematic illustration of TRIM18 serving as a negative regulator in antiviral innate immunity against organ inflammations induced by RNA and DNA viruses. In WT macrophages after infections with DNA and RNA viruses, TRIM18 interacts with PPM1A and induces its K63-linked ubiquitination for maintaining stability of PPM1A, thereby further dephosphorylating TBK1 for its inactivation and blocking the interactions of TBK1 with its upstream adaptors STING and MAVS leading to dramatic reduction of type I IFN, which promotes viral myocarditis and more massive inflammations in lung and brain. In contrast, in TRIM18 KO macrophages post infections with DNA and RNA viruses, PPM1A could not maintain its stability without TRIM18, and loss its strong ability to inactivate TBK1 resulting in significant induction of type I IFN, which restricts viral myocarditis, inflammations in lung and brain

Fig 4: Lack of palmitate (PA) response in hepatocytes of MD2 knockout mice is through suppressed TBK1.(A) Primary hepatocytes isolated from WT mice and MD2KO mice were treated with 200 µM PA for 1 h. Levels of p-TBK1 were determined by immunoblotting. GAPDH was used as loading control. Densitometric quantification is shown in lower panel. (B) Liver tissue lysates of WT and MD2KO mice fed a high-fat diet (HFD) for 16 weeks were probed for p-TBK1. GAPDH was used as loading control. Densitometric quantification is shown in right panel. (C) Immunoprecipitation of AMPK was performed in hepatocytes from WT and MD2KO mice after exposure to 200 µM PA for 1 h. Levels of TBK1 were detected by immunoblotting. (D) Primary hepatocytes from WT mice were pretreated with 20 µM GSK8612 (GSK) for 1 h before exposure to 200 µM PA for 1 h. Levels of p-TBK1 and p-AMPK were detected. GAPDH was used as loading control. (E) mRNA levels of Srebp1 in cells pretreated with 20 µM GSK for 1 h before exposure to 200 µM PA for 48 h. Data normalized to Actb. (F) AML-12 cell line was exposed to 200 µM PA for 1 h. Levels of p-TBK1 were detected. GAPDH was used as loading control. Densitometric quantification is shown on right. (G and H) AML-12 cells were pre-treated with GSK for 1 h and then challenged with PA for 1 h. TBK1-AMPK complex was examined by immunoprecipitation (G). Whole cell lysates were probed for p-TBK1 and p-AMPK (H). (I and J) AML-12 cells were pretreated with 20 µM GSK for 1 h before exposure to 200 µM PA for 48 h. mRNA levels of Srebp1 (I) and sterol regulatory element binding protein 1 (SREBP1)-target genes (J) were measured. Data normalized to Actb. (K) AML-12 cells were treated as indicated in panel I. Lipid accumulation was detected by oil red O staining (scale bar = 50 µm). Representative images are shown in panel K. Quantification of lipid area is shown in lower panel. In all panels, n = 3 independent experiments; data are shown in mean ± SEM; *p < .05, **p < .01 and ***p < .001]

Fig 5: TRIM18 induces ubiquitination of PPM1A by K63 linkage. a Immunoblot (IB) analysis of TRIM18 (fourth blot), PPM1A (fifth blot) and ß-actin (bottom blot) in WT and TRIM18 KO BMDM, the abundance (top blot), total ubiquitination (second blot), and K63-mediated ubiquitination (third blot) of PPM1A in those cells, infected for 4 h with CVB3 or adenovirus at an MOI of 5, assessed after immunoprecipitation with anti-PPM1A antibody. b Immunoblot analysis (with anti-Myc) of the abundance (top), total ubiquitination (second blot), and K63-linked ubiquitination (third blot) of Myc-tagged PPM1A in HEK293T cells transfected with empty vector or expression vector for full length HA-tagged TRIM18 (HA-T18 full), truncation T18-?SPRY (losing binding site of PPM1A), assessed after immunoprecipitation with anti-Myc antibody; immunoblot analysis of whole-cell lysates with anti-HA (fourth blot), anti-Myc (fifth blot) and anti-ß-actin (bottom). c Immunoblot analysis (with anti-Myc) of the abundance (top), total ubiquitination (second blot), and K63-linked ubiquitination (third blot) of Myc-tagged PPM1A in HEK293T cells transfected with HA-TRIM18 and Myc-PPM1A wild-type (WT) and its mutations including K9RK12R, K118RK119R, K156R, K296R, K303RK304R, and K354R, assessed after immunoprecipitation with anti-Myc antibody; immunoblot analysis of whole-cell lysates with anti-Myc (fourth blot), anti-HA (fifth blot) and anti-ß-actin (bottom). d Luciferase assay in HEK293T cells transfected with IFN-ß reporter, Flag-tagged plasmid expressing TBK1 along with Myc-tagged empty vector or PPM1A wild-type (WT) and its mutations including K9RK12R, K118RK119R, K156R, K296R, K303RK304R, and K354R. Renilla luciferase RL-TK was used as the internal control. e Immunoblot analysis (with anti-Myc) of the abundance (top), and K63-linked ubiquitination (second blot) of Myc-tagged PPM1A in HEK293T cells transfected with HA-TRIM18, Flag-TBK1 and Myc-PPM1A wild-type (WT) and its ubiquitination losing mutant K9/12/296/354R, assessed after immunoprecipitation with anti-Myc antibody; immunoblot analysis of whole-cell lysates with anti-phosphorylated (p-) TBK1 (third blot), anti-TBK1 (fourth blot) and anti-ß-actin (bottom). f Luciferase assay in HEK293T cells transfected with IFN-ß reporter, Flag-tagged plasmid expressing TBK1 along with Myc-tagged empty vector or PPM1A wild-type (WT) and its ubiquitination losing mutant K9/12/296/354R. Renilla luciferase RL-TK was used as the internal control. NS, not significant (p > 0.05), ***p < 0.001, and p value was calculated by unpaired two-tailed Student’s t test. The position of protein markers (shown in kDa) is indicated on the right. Data are representative of three independent experiments