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).