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FIGURE 1 a Workflow used in our study to enrich and profile ADP-ribosylated and ADP-riboxanated proteins, which is adapted from our previous publication (Jin et al., 2023). b-c OspC1 fromS. flexneri were expressed in 293T cells, and ADP-ribosylated or ADP-riboxanated proteins were isolated from cell lysates using eAf1521-conjugated beads. The samples and cell lysates were separated by SDS-PAGE, and then subjected to immunoblotting (b), or coomassie blue staining and LC-MS/MS analysis (c), respectively. d HERC5-Flag was co-expressed with wild-type (WT) OspC1 or catalytically dead mutant (MUT) OspC1 E187A/H322A in 293T cells. Cells were collected and lysed and subjected to immunoblotting with indicated antibodies, or immunoprecipitation with Flag antibodies, followed by immunoblotting with indicated antibodies. e HERC5-Flag isolated from WT or MUT OspC1-overexoressing 293T cell lysates were separated by SDS-PAGE and then subjected to coomassie blue staining.
FIGURE 2 a-b LC-MS/MS analysis of ADP-riboxanated HERC5-Flag in collision-induced dissociation (CID) mode (b) or in high energy collision dissociation (HCD) mode. Tandem mass (MS/MS) spectrums indicated that HERC5 was ADP-roboxanated at Arg44 and matched the fragmentation pattern of the modified peptide with many ADP-riboxanation-specific diagnostic ions and neutral loss fragments.c Extracted ion chromatograms of the reference peptide, unmodified peptide, and modified peptide with peak intensities.
FIGURE 3 a MS/MS spectrum matching the ADP-ribosylated peptide of HERC5 with ADP-ribosylation-specific diagnostic ions. bExtracted ion chromatograms of the ADP-ribosylated or ADP-riboxanated peptide. c Schematic illustration of the reaction of ADP-ribosylation and ADP-riboxanation adjusted from our previous publication (Jin et al., 2023).
FIGURE 4 a 293T cells were treated with IFN-β for indicated times and then harvested and lysed, followed by immunoblotting with indicated antibodies. b ISG15-conjugating system including Ube1L (E1), UBCH8 (E2), ISG15 were co-expressed with HERC5 (E3) or HERC5 and USP18 in 293T cells. Cells were collected and lysed for immunoblotting with indicated antibodies. c 293T cells overexpressing ISG15-conjugating system with WT or MUT OspC1 were harvested and lysed for immunoblotting with indicated antibodies.d-e Flag-tagged HERC5 (d) or RIG-I (e) were overexpressed with ISG15-conjugating system in the presence of WT or MUT OspC1 and isolated with anti-Flag agarose and then immunoblotted with anti-ISG15 antibody.
FIGURE 5 A working model for regulation of RIG-I signaling byShigella flexneri effector OspC1-catalyzed HERC5 ADP-riboxanation. ADP-riboxanation of HERC5 promotes its E3 ISG15 ligase activity and then enhances the ISGylation of RIG-I, which results in the decrease of unmodified and functional RIG-I and thereby downregulation of RIG-I-mediated anti-bacterial defence.
FIGURE S1 Tandem mass (MS/MS) spectrums indicated that HERC5 was ADP-roboxanated at Arg663 and matched the fragmentation pattern of the modified peptide with ADP-riboxanation-specific diagnostic ions.