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. 2022 Mar 16;119(12):e2116776119. doi: 10.1073/pnas.2116776119

Fig. 5.

Fig. 5.

The interaction of IpaH1.4 LRR with HOIP RING1 or HOIL-1L UBL is essential for the in vitro ubiquitination of HOIP or HOIL-1L as well as the suppression of NF-κB activation in cells. (A and B) In vitro ubiquitination assays showing the abilities of the wild-type IpaH1.4 (A) and the IpaH1.4 R157A mutant (B) to assemble K48-type Ub chains on the three subunits of LUBAC. To eliminate the potential ubiquitination events induced by HOIP or HOIL-1L, the catalytic-dead HOIP (480 to 1,072) C885A and HOIL-1L C460A mutants are used in these assays. Notably, the IpaH1.4 R157A mutant, which is unable to interact with HOIP RING1, almost loses the ability to assemble K48-type Ub chains on HOIP; IB, immunoblot; HA, hemagglutinin; WT, wild-type. (C) In vitro ubiquitination assays showing the abilities of the wild-type IpaH1.4 (Left), the IpaH1.4 R215E mutant (Middle), and the IpaH1.4 F238E/N240D mutant (Right) to mediate the poly-ubiquitination of HOIL-1L. Notably, the R215E and F238E/N240D mutants of IpaH1.4, both of which are unable to interact with HOIL-1L UBL, cannot mediate the poly-ubiquitination of HOIL-1L. (D) NF-κB reporter dual-luciferase assay using overexpressed LUBAC together with the wild-type IpaH1.4 or different IpaH1.4 variants. All luciferase activities are normalized to that of the control cells. Error bars denote the SD between three replicates. An unpaired Student's t test analysis was used to define a statistically significant difference, and the stars indicate the significant differences between the indicated bars (**, P ≤ 0.01; ***, P ≤ 0.001). (E) Representative fluorescent microscopy images of cultured HeLa cells infected with the wild-type S. flexneri M90T strain, the ipaH1.4/ipaH2.5 double-knockout S. flexneri (ΔipaH1.4/ΔipaH2.5) strain, or a S. flexneri (ΔipaH1.4/ΔipaH2.5) strain recomplemented with the wild-type IpaH1.4 or different IpaH1.4 mutant (R175A, C368A, R175A/C368A, 4M, and 4M/C368A) and stained with a specific antibody to the p65 subunit of NF-κB (red) and DAPI (blue) to show bacteria and nuclei of cells. For comparison, DAPI and p65 fluorescence intensity values in a cross-section of a selected cell (indicated with a white line) are shown in graphs on the Right. (F) Statistical results related to the nuclear translocation of p65 in HeLa cells infected with different S. flexneri strains in E. Bar graphs represent results from three independent experiments presented as the mean ± SD of >70 analyzed cells. An unpaired Student's t test analysis was used to define a statistically significant difference, and the stars indicate the significant differences between the indicated bars (**, P ≤ 0.01; ***, P ≤ 0.001); ns, not significant.