Carboxyl-terminus of HSC70-interacting protein (CHIP, also known as STIP1 homology and U-Box containing protein 1, STUB1) is a cochaperone E3 ligase. STUB1 (CHIP) contains three tandem repeats of tetratricopeptide (TPR) motifs and a C-terminal U-box domain separated by a charged coiled-coil region (Paul I & Ghosh MK 2014). STUB1 functions as a negative co-chaperone for the HSP90/HSP70 chaperone to regulate protein quality control by targeting unfolded or misfolded proteins for proteasomal degradation. Structural studies suggest that STUB1 functions as a homodimer (Zhang M et al. 2005). In addition, STUB1 (CHIP) also targets many mature proteins for ubiquitination and degradation or degradation-independent regulation (Paul I & Ghosh MK 2014). STUB1 has been shown to affect apoptotic cell death by negatively regulating a variety of tumor suppressive factors (Ahmed SF et al. 2012; Esser C et al. 2005). STUB1 (CHIP) has been also implicated in down-regulation of necroptosis via targeting receptor-interacting serine/threonine protein kinase 1 (RIPK1) and RIPK3 (Seo J et al. 2016). STUB1 deficiency in mouse embryonic fibroblasts (MEF), mouse fibroblasts (L929) and human colorectal adenocarcinoma (HT-29) cells exhibited higher levels of RIPK1 and RIPK3 expression, resulting in increased sensitivity to necroptosis induced by TNFα (Seo J et al. 2016). Supporting these findings, in vivo studies demonstrated that the inflammatory and lethal phenotypes of Chip−/− mice were rescued by crossing with Ripk3 knockout mice (Seo J et al. 2016). Coimmunoprecipitation analysis revealed interactions between STUB1 and RIPK1 or RIPK3 (Seo J et al. 2016). K571, K604, and K627 as ubiquitination lysine sites of RIPK1 were detected by mass spectrometry (Mollah S et al. 2007; Kim W et al. 2011). Mutagenesis analyses of RIPK1 demonstrated that STUB1 (CHIP)-mediated K48-ubiquitination on K571, K604, and K627 of RIPK1 is essential for the lysosome co-localization and degradation of RIPK1 upon co-expression of tagged proteins in human non–small-cell lung cancer H1299 cells (Seo J et al. 2016). Similarly, STUB1 ubiquitinated RIPK3 inducing lysosome-dependent destabilization of RIPK3 (Seo J et al. 2016). Further, RIPK1 I539D and RIPK3 V460P, that do not form a hetero-oligomeric amyloid signaling RIPK1:RIPK3 complex, also showed lysosomal localization upon ectopic expression of STUB1 in H1299 cells suggesting that STUB1 targets nascent forms of RIPK3 and RIPK1. The data suggest that STUB1 negatively regulates RIPK1 & RIPK3-mediated necroptosis via K48-linked ubiquitinitaion and lysosome-dependent destabilization of RIPK1 and RIPK3.

RIPK1 functions as a key regulator of the TNF receptor 1 (TNFR1) signaling, which is activated upon binding of TNF-α to TNF receptor 1 (TNFR1). Activation of TNFR1 results in the sequential formation of several signaling complexes (Micheau O and Tschopp J 2003; Walczak H 2011). The rapidly forming complex-I (the TNFR1 signaling complex) is assembled at the receptor’s cytoplasmic tail and consists of TNFR1, TRADD (TNFR1-associated death domain), TRAF2 (TNF receptor associated factor-2), RIPK1, and E3 ubiquitin (Ub) ligases BIRC2, BIRC3 (cIAP1/2, cellular inhibitor of apoptosis) and LUBAC (linear ubiquitin chain assembly complex) (Micheau O and Tschopp J 2003; Yuan J et al. 2019). Within this complex, RIPK1 and other proteins are rapidly conjugated with Ub chains by various E3 ligases (Micheau O and Tschopp J 2003; Walczak H 2011; Yuan J et al. 2019; Roberts JZ et al. 2022). The ubiquitination/deubiquitination status of RIPK1 determines cell fate downstream of the TNFR1 signaling complex (Yuan J et al. 2019; Roberts JZ et al. 2022). The conjugation of K63-linked Ub chains by BIRC2/3 or Met1-linked Ub chains by LUBAC, have been shown to promote RIPK1-dependent pro-survival NF-kappa-B signaling while inhibiting RIPK1 kinase-mediated apoptosis and necroptosis. E3 ubiquitin ligase activity of MIB2 also protects cells from the RIPK1-mediated cell death (Feltham R et al. 2018). Deubiquitination of RIPK1 abolishes its ability to activate NF-kappa-B upon TNF-α stimulation and leads to the formation of the cytosolic complex IIa, TRADD:TRAF2:RIPK1:FADD:caspase-8, which activates apoptosis. In addition, RIPK1 also interacts with RIPK3 and MLKL to form complex IIb, which activates necroptosis (Micheau O and Tschopp J 2003; Yuan J et al. 2019). In these cell death-inducing complexes, RIPK1 activity is also regulated by ubiquitination (Amin P et al. 2018; de Almagro M et al. 2015). Finally, E3 ligase activity of STUB1 targets RIPK1 and RIPK3 for lysosome-dependent degradation suppressing necroptosis (Seo J et al. 2016).

This Reactome event describes STUB1-mediated K48-linked ubiquitination of RIPK1 at K571, K604, and K627 downstream of TNFR1.