de Oliveira Mann CC , Orzalli MH , King DS , Kagan JC , Lee ASY , Kranzusch PJ .

             regulation of type I interferon-mediated signaling pathway
             positive regulation of innate immunity memory response

	Figure 1STING C-Terminal Modules Control the Balance of Downstream IRF3 and NF-κB Signaling (A) Reconstitution of the cGAS-STING pathway in human cells using a phylogenetically diverse panel of vertebrate STING alleles. Luciferase reporters were used to monitor cGAS-STING dependent interferon β (IFNβ; blue) and NF-κB (orange) responses. Species shown are as follows: 1, human (Homo sapiens); 2, marmoset (Callithrix jacchus); 3, mouse (Mus musculus); 4, cat (Felis catus); 5, seal (Leptonychotes weddellii); 6, cattle (Bos taurus); 7, boar (Sus scrofa); 8, bat (Rousettus aegyptiacus); 9, manatee (Trichechus manatus latirostris); 10, Tasmanian devil (Sarcophilus harrisii); 11, ostrich (Struthio camelus australis); 12, crested ibis (Nipponia nippon); 13, lizard (Anolis carolinensis); 14, turtle (Chelonia mydas); 15, western clawed frog (Xenopus tropicalis); 16, African clawed frog (Xenopus laevis); 17, coelacanth (Latimeria chalumnae); 18, salmon (Salmo salar); 19, zebrafish (Danio rerio); and 20, ghost shark (Callorhinchus milii). (B) Schematics of STING domain organization (TM, transmembrane domain; CDN, cyclic dinucleotide binding domain; CTT, C-terminal tail). Cellular reporter assay as in (A), mapping the motif responsible for enhanced NF-κB signaling to the CTT of zebrafish STING. (C) Cellular reporter assay and schematics as in (B), comparing downstream signaling outputs of human STING with human STING containing the zebrafish STING CTT sequence. The zebrafish STING CTT is sufficient to enhance NF-κB signaling. Cellular reporter assay data are representative of at least three independent experiments. Data are plotted with error bars representing the SD of the mean.
	Figure 2. The Zebrafish STING Contains a Unique CTT Module that Boosts NF-κB Signaling(A) Schematics of STING chimera CTT constructs and sequences of human STING T356-S379 and zebrafish STING P363-N398 aligned using Jalview and with three highlighted signaling motifs mapped for type I interferon (dark and light blue) and NF-κB (orange). The human STING residues S366 and L374 required for interferon signaling are marked in red.(B) Reconstitution of the cGAS-STING pathway in human cells using different human-zebrafish STING chimera constructs. Interferon (blue) and NF-κB (orange) reporter activity was measured as in Figure 1A. All STING CTT chimeras consist of a human core domain with three variable CTT modules (dark blue, light blue, and orange) from human and zebrafish STING, respectively (constructs CTT-a to CTT-o).Cellular reporter assay data are representative of at least three independent experiments. Data are plotted with error bars representing the SD of the mean.
	Figure 3. The Zebrafish STING CTT Module Directly Recruits TRAF6 to Activate NF-κB Signaling(A) Schematics of zebrafish STING and sequence logo of the highlighted STING CTT signaling factor recruitment motifs of mammals compared with ray-finned fish species (9 amino acids [aa]; 6 aa for interferon signaling and an additional 7 aa in ray-finned fish for NF-κB signaling). Amino acids are numbered according to the human STING sequence.(B) Reporter assay for IFNβ (blue) or NF-κB (orange) using human and zebrafish STING in TBK1 knockout (KO) HEK293T cells. IFNβ signaling is lost in TBK1 KO cells. However, the NF-κB signaling responses for human and zebrafish STING persist in TBK1 KO cells.(C) Reporter assay as in (B), performed in TRAF6 KO HEK293T cells. All zebrafish STING-dependent NF-κB signaling is abolished in TRAF6 KO cells.(D) Reporter assay as in (B), performing alanine scan mutagenesis of the TRAF6 binding motif in the zebrafish STING CTT chimera containing a human STING core domain. Consistent with other TRAF6 recruitment motifs, only the P0 position is essential for STING-TRAF6 complex formation.(E) Cellular assay as in (B), monitoring STING-dependent signaling in the presence of alternative TRAF6 alleles. Endogenous TRAF6 was removed (TRAF6 KO), and signaling was reconstituted with plasmids encoding alternative TRAF6 and TRAF3 alleles as indicated. Disruption of the TRAF6 E3 ligase domain (L74H) or expression of only the TRAF domain prevents zebrafish STING-dependent NF-κB signaling.Cellular reporter assay data are representative of at least three independent experiments. Data are plotted with error bars representing the SD of the mean.
	Figure 4. Structural Basis of Zebrafish STING-TRAF6 Complex Formation(A) Schematics of zebrafish TRAF6 domain organization (RING domain, zinc fingers, coiled coil [CC], and TRAF domain) and the co-crystal structure of the zebrafish TRAF6 C-domain (light gray) in complex with the zebrafish STING CTT NF-κB motif (orange) at 1.4Å. The STING CTT peptide 2Fo–Fc electron density is shown in gray and contoured at 1.0 σ.(B) Interaction map showing the interface between TRAF6 and the zebrafish STING CTT peptide. Main-chain hydrogen bonds between the STING CTT (orange) and the TRAF6 β7 residues (light gray) are shown as dotted lines.(C) Comparison of TRAF6 (light gray) interactions with aromatic or acidic residues at peptide position P3 (orange). STING has an acidic D383 at position P3 that is recognized by an arginine residue at the bottom of the pocket (human TRAF6 R466, zebrafish TRAF6 R487), whereas RANK (PDB: 1LB5) has an aromatic residue, Y349, at the same position that interacts with a distinct arginine at the top of the pocket (human TRAF6 R392, zebrafish TRAF6 R416).
	Figure 5. Acquisition of the STING TRAF6 Module Remodels Downstream Immune Responses(A) Schematic representing generation of mouse macrophage cell lines with different STING gene alleles.(B) Western blot analysis of STING expression in different mouse macrophage cell lines compared with the RPS19 loading control. Data are representative of three biological experiments.(C) Heatmap of reads per kilobase of transcript per million mapped reads (RPKM) of 100 differentially upregulated genes in mouse macrophage cell lines: control, mouse STING (mSTING) ΔCTT, mSTING, and mSTING zebrafish C-terminal tail (zfCTT). Acquisition of the zebrafish STING CTT enhances the STING-dependent interferon response and additionally activates alternative NF-κB responses.(D) Venn diagram depicting upregulated genes, the normal mouse STING response, and the alternative mSTING zfCTT response.(E) Visualization of RNA sequencing (RNA-seq) gene coverage and transcript levels for the selected differentially expressed genes Ifnb, Il12b, nos2, and ccl12 in macrophage cell lines: control, mSTING ΔCTT, mSTING, and mSTING zfCTT using an integrative genomics viewer (IGV) (Robinson et al., 2011).(F) Transcript-level analysis of selected genes by qRT-PCR. qRT-PCR data are representative of three independent experiments.Data are plotted with error bars representing the SD of the mean.

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