Mind bomb 1 (MIB1) and MIB2 belong to RING-type E3 ubiquitin (Ub) ligases. Both MIB1 and MIB2 are known to regulate NOTCH signaling through targeting the NOTCH ligands Delta and Jagged for ubiquitination and internalization (reviewed in Dutta D et al 2022). MIB1 and MIB2 have been also implicated in regulation of NF-kappa-B-mediated inflammatory responses, interferon induction and cell death (Zhang L & Gallagher PJ 2009; Stempin CC et al. 2011; Li S et al 2011; Ye JC et al. 2014; Feltham R et al. 2018; Uematsu A et al. 2019). MIB2 has been implicated in regulation of the tumor necrosis factor alpha (TNFα)-induced pathway. Ligation of TNFα to TNF receptor 1 (TNFR1) induces the sequential formation of several signaling complexes to promote either cell survival (complex I) or cell death (complex II) (Micheau O and Tschopp J 2003; Walczak H 2011; Yuan J et al. 2019). The assembly of these complexes is tightly regulated by proteolysis, ubiquitination and phosphorylation of receptor-interacting serine/threonine protein kinase 1 (RIPK1) and other components of the TNFα signaling pathway (reviewed in Yuan J et al. 2019; Varfolomeev E & Vucic D 2022). RIPK1 functions as a key regulator of both cell survival and cell death (reviewed in Ju E et al. 2022). Enzymatically inactive polyUb-bound RIPK1 serves as a scaffold in the membrane-bound TNFR1 signaling complex (complex I) contributing to activation of mitogen-activated protein kinase (MAPK) and NF-kappa-B signaling pathways, which regulate expression of pro-survival and inflammatory genes. Deubiquitination of RIPK1 negatively regulates TNFα-induced pro-survival responses while promoting RIPK1-mediated cell death (reviewed in Ju E et al. 2022). Several deubiquitinating peptidases regulate cytotoxic potential of RIPK1 downstream of TNFR1, including Ub carboxyl-terminal hydrolase CYLD, which is capable of cleaving K63-linked and Met1-linked polyUb chains. The E3 Ub ligase activity of MIB2 controls the TNFα:TNFR1 pathway by targeting both RIPK1 and CYLD (Feltham R et al. 2018; Uematsu A et al. 2019). MIB2 directly binds and conjugates different types of polyUb chains to RIPK1 within the TNFR1 signaling complex I (Feltham R et al. 2018). MIB2-mediated ubiquitination of RIPK1 inhibits TNFα-induced cell death (Feltham R et al. 2018). Direct interaction of MIB2 with CYLD was detected by glutathione S-transferase (GST) pull down assay coupled with mass spectrometry analysis using GST-CYLD fusion protein as a bait in TNFα-stimulated human embryonic kidney 293T (HEK293T) cells (Rajan N et al. 2014). Co-immunoprecipitation (Co-IP) assay identified CYLD:MIB2 interaction upon co-expression of tagged proteins in HEK293T cells (Uematsu A et al. 2019). Co-IP also detected interaction between endogenous CYLD and MIB2 in HEK293T cells (Rajan N et al. 2014; Uematsu A et al. 2019). Immunofluorescence assay revealed that both CYLD and MIB2 were co-localized in the cytoplasm of HeLa cells (Uematsu A et al. 2019). Mutagenesis studies showed that the third cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of CYLD and the ankyrin repeat region of MIB2 are necessary and sufficient for CYLD:MIB2 interaction (Uematsu A et al. 2019). MIB2-mediated K48-linked ubiquitination of CYLD targets CYLD for proteasomal degradation thus preventing CYLD-mediated deubiquitination of RIPK1 and RIPK1-dependent cell death (Uematsu A et al. 2019). These data suggest that the E3 ligase activity of MIB2 suppresses the TNF-induced cell death by attaching polyUb chains to CYLD (Uematsu A et al. 2019)

This Reactome event describes binding of MIB2 to CYLD in the cytosol. Both MIB2 and CYLD are recruited to the membrane-bound TNFR1 complex, suggesting that their interaction may also occur within the complex I (not shown here).