Identification of HERC5 as a host substrate of S. flexneri effector OspC1
To identify host substrates of S. flexneri effector OspC1, we overexpressed empty vector (EV) or wild-type (WT) OspC1 in 293T cells. ADP-riboxanated and ADP-ribosylated proteins were isolated with eAF1521-conjugated beads from EV or WT OspC1-transfacted cell lysates and then digested with trypsin and subjected to mass spectrometry (MS) analysis as described in our previous publication (Figure 1a; Jin et al., 2023). As we expected, ADP-riboxanation or ADP-ribosylation signals were significantly stronger, and more proteins were enriched in WT group than in EV group (Figure 1b, c). In the proteomic dataset, we noticed an extremely familiar protein HERC5, which we have studied before (Jin et al., 2021), was detected in the WT group other than the EV group (Supplementary Table 1). Known that ISG15-conjugating system including HERC5 are highly inducible upon type I interferon stimulation or bacterial infection, we tried to verify HERC5 as a host substrate ofS. flexneri effector OspC1 in the overexpression system with WT OspC1 or its catalytic dead mutant (MUT) OspC1E187A/H322A. Fortunately, we confirmed that HERC5 is indeed a host substrate of S. flexneri effector OspC1 (Figure 1d).