Co-immunoprecipitation assay showed that TRIM31 interacted with NLRP3 in mouse and human cells (Song H et al. 2016; Huang P et al. 2020). The E3 ubiquitin-protein ligase TRIM31 was found to mediate the K48-linked ubiquitination of NLRP3 upon co-expression of NLRP3, TRIM31 with HA-tagged ubiquitin in human embryonic kidney 293T (HEK293T) cells (Song H et al. 2016). TRIM31 promoted proteasomal degradation of NLRP3 in HEK293T cells. Similar results were obtaned in mouse peritoneal macrophages (Song H et al. 2016). Mutagenesis analysis showed that the N-terminal RING domain of TRIM31 is crucial for its E3 ligase activity and TRIM31-mediated K48-linked ubiquitination and degradation of NLRP3. Further, TRIM31 truncated mutants with deleted RING domain lost the ability to inhibit caspase-1 cleavage, compared with WT TRIM31 in THP-1 cells (Song H et al. 2016). Lys-Arg site-directed mutagenesis suggests that NLRP3 is ubiquitinated by TRIM31 at K496 (Zhao W et al. 2020). Ubiquitin-conjugating Enzyme E2D (UBE2D1 or UbcH5A) facilitated K48-linked polyubiquitination of NLRP3 by TRIM31 in vitro (Song H et al. 2016). AKT-mediated phosphorylation of NLRP3 at S5 was shown to inhibit TRIM31-mediated ubiquitination of NLRP3 (Zhao W et al. 2020). These data suggest that the E3 ubiquitin ligase activity of TRIM31 negatively regulates the NLRP3 inflammasome activation through promoting K48-linked polyubiquitination of NLRP3 at K496 and triggering proteasome-mediated degradation of NLRP3. However, one study reported that silencing the Trim31 gene in mouse bone marrow-derived macrophages (BMDM) did not affect LPS-primed, ATP-stimulated production of IL-1β suggesting that TRIM31 may not function as the E3 ubiquitin ligase that targets NLRP3 (Tang J et al. 2020).

This Reactome event describes TRIM31-mediated polyubiquitination of NLRP3 at K496 in the presence of Ub-conjugating E2 enzyme UBE2D1.