STUB1 ubiquitinates RIPK1 at K571, K604, K627

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

羧基端HSC70相互作用蛋白（CHIP，亦称STIP1同源和U-Box含蛋白1，STUB1）是一种辅助性E3连接酶。STUB1（CHIP）含有三个串联的四肽重复结构（TPR）基序和一个由带电的螺旋卷曲区域分隔的C端U-Box结构域（Paul I & Ghosh MK 2014）。STUB1作为HSP90/HSP70辅助蛋白的负性共辅助因子，通过靶向未折叠或错误折叠的蛋白质，以调节蛋白质质量控制，并使其进行蛋白酶体降解。结构研究表明，STUB1以同源二聚体的形式发挥作用（Zhang M et al. 2005）。此外，STUB1（CHIP）还靶向许多成熟蛋白质进行泛素化、降解或降解无关的调节（Paul I & Ghosh MK 2014）。STUB1已被证明通过负性调节多种肿瘤抑制因子影响细胞凋亡性细胞死亡（Ahmed SF et al. 2012; Esser C et al. 2005）。STUB1（CHIP）还与通过靶向受体相互作用丝氨酸/苏氨酸蛋白激酶1（RIPK1）和RIPK3下调细胞焦亡有关（Seo J et al. 2016）。在鼠胚胎成纤维细胞（MEF）、鼠成纤维细胞（L929）和人类结直肠腺癌（HT-29）细胞中，STUB1的缺乏导致RIPK1和RIPK3表达水平升高，从而增加对TNFα诱导的细胞焦亡的敏感性（Seo J et al. 2016）。支持这些发现，体内研究表明，通过与Ripk3敲除小鼠杂交，Chip−/−小鼠的炎症和致死表型得到挽救（Seo J et al. 2016）。共免疫沉淀分析揭示了STUB1与RIPK1或RIPK3之间的相互作用（Seo J et al. 2016）。通过质谱检测到RIPK1的K571、K604和K627作为泛素化赖氨酸位点（Mollah S et al. 2007; Kim W et al. 2011）。对RIPK1的突变分析表明，STUB1（CHIP）介导的RIPK1的K571、K604和K627的K48泛素化对于在人类非小细胞肺癌H1299细胞中与标记蛋白共表达时RIPK1的溶酶体共定位和降解是必需的（Seo J et al. 2016）。同样，STUB1泛素化RIPK3诱导其溶酶体依赖性不稳定（Seo J et al. 2016）。进一步地，在H1299细胞中异位表达STUB1后，RIPK1 I539D和RIPK3 V460P，它们不形成异源寡聚的淀粉样信号RIPK1:RIPK3复合物，也表现出溶酶体定位，这表明STUB1靶向RIPK3和RIPK1的初生形式。数据表明，STUB1通过K48连接的泛素化和RIPK1和RIPK3的溶酶体依赖性不稳定，负性调节RIPK1 & RIPK3介导的细胞焦亡。RIPK1作为TNF受体1（TNFR1）信号通路的关键调节因子，在TNF-α与TNFR1结合后激活。TNFR1的激活导致多个信号复合物的顺序形成（Micheau O and Tschopp J 2003; Walczak H 2011）。快速形成的复合物-I（TNFR1信号复合物）在受体的胞质尾端组装，由TNFR1、TRADD（TNFR1相关死亡域）、TRAF2（TNF受体相关因子-2）、RIPK1和E3泛素连接酶BIRC2、BIRC3（cIAP1/2，细胞凋亡抑制因子）和LUBAC（线性泛素链组装复合物）组成（Micheau O and Tschopp J 2003; Yuan J et al. 2019）。在此复合物中，RIPK1和其他蛋白质通过各种E3连接酶迅速与泛素链共价连接（Micheau O and Tschopp J 2003; Walczak H 2011; Yuan J et al. 2019; Roberts JZ et al. 2022）。RIPK1的泛素化/去泛素化状态决定了TNFR1信号复合物下游的细胞命运（Yuan J et al. 2019; Roberts JZ et al. 2022）。BIRC2/3连接的K63链泛素化或LUBAC连接的Met1链泛素化已被证明可促进RIPK1依赖性存活NF-kappa-B信号通路，同时抑制RIPK1激酶介导的凋亡和细胞焦亡。MIB2的E3泛素连接酶活性也保护细胞免受RIPK1介导的细胞死亡（Feltham R et al. 2018）。RIPK1的去泛素化消除了其在TNF-α刺激下激活NF-kappa-B的能力，并导致细胞质复合物IIa的形成，即TRADD:TRAF2:RIPK1:FADD:caspase-8，从而激活凋亡。此外，RIPK1还与RIPK3和MLKL相互作用，形成复合物IIb，从而激活细胞焦亡（Micheau O and Tschopp J 2003; Yuan J et al. 2019）。在这些细胞死亡诱导的复合物中，RIPK1活性也受泛素化的调节（Amin P et al. 2018; de Almagro M et al. 2015）。最后，STUB1的E3连接酶活性靶向RIPK1和RIPK3进行溶酶体依赖性降解，抑制细胞焦亡（Seo J et al. 2016）。本Reactome事件描述了STUB1介导的TNFR1下游RIPK1的K571、K604和K627的K48连接泛素化。