Fig 1: Ppia was mono-ADP ribosylated at the E140 residue by PARP3 to promote macrophage inflammation. A Schematic diagram of the homology of Ppia in mice, rats and humans. B Schematic diagram of the Ppia mutation sites. C Comparison of the effects of the D9A, E120A and E140A mutants on promoting inflammation (n = 3). The mRNA levels of IL-1β, IL-6 and Ppia were detected by qPCR. D RAW264.7 cells were transfected with the Ppia-WT and Ppia-E140A plasmids, and their ability to stimulate inflammation was tested by determining the phosphorylation status of p65 (n = 3). Protein levels of phosphorylated p65 were measured by western blot. E The modification ability of the purified Ppia-WT and Ppia-E140A mutant proteins in the in vitro system was tested by western blot (n = 3). F Mono-ADP-ribosylation on Ppia E140 was confirmed by immunoprecipitation of Ppia and detection with a mono-ADPr antibody (n = 3). G The purified Ppia-WT and Ppia-E140A proteins were added to RAW264.7 cell cultures to detect the expression of IL-1β and IL-6 by qPCR and (H) phosphorylation of p65 by western blot (n = 3). I The secretion of Ppia was influenced by mono-ADP ribosylation and was detected by ELISA (n = 7). J The purified Ppia-WT and Ppia-E140A proteins were added to MPM cell cultures to detect the expression of IL-1β and IL-6 by qPCR (n = 3). K Schematic diagram of the mechanism by which PAPR3 mono-ADP ribosylate Ppia to stimulate the NF-κB pathway and activate inflammation in macrophages. The data were expressed as the means ± standard deviations. One-way ANOVA was used for statistical analysis in (C-J). *p < 0.05, **p < 0.01, ***p < 0.001 or ****p < 0.0001 were considered significant
Fig 2: Ppia mediated PARP3 to promote inflammation. A The proinflammatory effect of PARP3 requires the mediation of Ppia. The expression of IL-1β was inhibited with the knockdown of Ppia (n = 3). B and the phosphorylation of p65 was also suppressed. (n = 3). C The presence of the PARP3 inhibitor ME0328 inhibited the increase in IL-6 and D the phosphorylation of p65 induced by Ppia overexpression (n = 3). E LPS-induced IL-1β & IL-6 expression (F) and p65 phosphorylation were inhibited by transfecting the siRNA of Ppia (n = 3). The mRNA levels of IL-1β, IL-6, PARP3 and Ppia were detected by qPCR and protein levels of phosphorylated p65 were measured by western blot. The data were expressed as the means ± standard deviations. One-way ANOVA was used for statistical analysis in (A-F). *p < 0.05, **p < 0.01, ***p < 0.001 or ****p < 0.0001 were considered significant
Fig 3: Ppia interacted with PARP3. A Schematic diagram of the screen for the candidate substrate of PARP3. B The top five proteins among the fifteen intersecting proteins were listed. These proteins were candidate substrates for PARP3. C The peptide map of the Ppia protein was presented. D Co-IP validated the interaction between PARP3 and Ppia
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