In addition, the linear polyubiquitination has been implicated in the NF-kappa-B activation. The linear ubiquitin chain assembly complex (LUBAC) ligase consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains (Kirisako T et al. 2006; Walczak H et al. 2012). IKBKG (NEMO), a regulatory component of the I\u03baB kinase (IKK) complex, is a substrate of LUBAC. LUBAC-mediated IKBKG ubiquitination enhances IKBKG interaction with the TNFR1 complex and stabilizes this protein complex to promote activation of NF-kappa-B (Haas TL et al. 2009).
Structural analysis revealed that NPL4 zinc finger 1 (NZF1) of HOIP can simultaneously bind both leucine zipper domains of NEMO (IKBKG) and ubiquitin and that both interactions are involved in the TNF alpha-mediated NF-kappa-B activation (Fujita H et al. 2014). In addition, NEMO (IKBKG) ubiquitination required RING-between-RING (RBR) domain of HOIL-1L (Smit JJ et al. 2013)", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q5VV17-1", "Q12933-1", "Q13107-1", "O75604-1", "O15111-1", "P98170-1", "Q13546-1", "P21580-1", "O14920-1", "Q13490-1", "Q13489-1", "Q6GQQ9-1", "Q9NQC7-1", "Q9UK80-1" ], "evidence": [ { "id": "R-HSA-5357776", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-5357776" } ] }, { "id": "R-HSA-5357831", "name": "CHUK, IKBKB phosphorylate CYLD at S418", "equation": "", "summary": "CYLD is a deubiquitinating enzyme (DUB) that removes K63-linked ubiquitin chains from a large number of key signaling molecules, including tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and RIPK1. CYLD knockdown in human embryonic kidney 293 cells and human cervical carcinoma HeLa cells resulted in constitutive ubiquitination of TRAF2 (Reiley W et al. 2005) At the same time stimuli-induced TRAF2 ubiquitination was associated with site-specific phosphorylation of CYLD, a molecular event that was shown to inhibit CYLD-mediated deubquitination of TRAF2 (Reiley W et al. 2005; Hutti JE et al. 2009). Phosphorylation of CYLD was detected in TNF-alpha-stimulated HEK293T and HeLa cells, in LPS-treated BJAB cells, a human B\u2011cell line, and in human Jurkat T cells after stimulation with mitogens (Reiley W et al. 2005). Phoshorylation of CYLD was found to depend on IKKgamma (IKBKG), since it was blocked in IKBKG-deficient Jurkat T cells (Reiley W et al. 2005). Transfection and in vitro kinase assays reveal that both IKKalpha (CHUK) and IKKbeta (IKBKB) are able to phosphorylate CYLD (Reiley W et al. 2005). The noncanonical IKK family member IKKepsilon (IKBKE) was also reported to phosphorylate CYLD at serine 418 inhibiting CYLD deubiquitinase activity. The phosphorylation of CYLD by IKKepsilon is thought to contribute to IKKepsilon-driven cell transformation (Hutti JE et al. 2009).", "cellular_location": "cytosol", "input_participant_list": [ "R-HSA-741420" ], "output_participant_list": [ "R-HSA-5212667" ], "enzyme_list": [ "O15111-1", "O14920-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-5357831", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-5357831" } ] }, { "id": "R-HSA-5357845", "name": "K63polyUb-RIPK1 is deubiquitinated", "equation": "", "summary": "K63-deubiquitination of RIP1 abolishes its ability to activate NF-kappa-B upon TNF-alpha stimulation and leads to the formation of the cytosolic caspase-8 containing complex II and subsequent apoptosis.", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q5VV17-1", "Q12933-1", "Q13107-1", "O75604-1", "P98170-1", "Q13546-1", "P21580-1", "Q13490-1", "Q13489-1", "Q6GQQ9-1", "Q9NQC7-1", "Q9UK80-1" ], "evidence": [ { "id": "R-HSA-5357845", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-5357845" } ] }, { "id": "R-HSA-5357860", "name": "TNF:TNFR1:TRADD:TRAF2:K63polyUb-RIPK1:BIRC2/3:LUBAC recruits TAK1 complex", "equation": "", "summary": "Polyubiquitinated RIPK1 binds to TAB2 resulting in the recruitment of the TAB2:TAK1 complex. The K63-linked ubiqitination on Lys377 of RIPK1 was shown to mediate the association of RIPK1 with the TAB2:TAK1 complex (Li H et al. 2006)", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q5VV17-1", "O43318-1", "Q12933-1", "Q13107-1", "O75604-1", "P98170-1", "Q13546-1", "P21580-1", "Q13490-1", "Q13489-1", "Q6GQQ9-1", "Q9NQC7-1", "Q9UK80-1" ], "evidence": [ { "id": "R-HSA-5357860", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-5357860" } ] }, { "id": "R-HSA-5357928", "name": "CLIP3 and CYLD bind TNF signaling complex", "equation": "", "summary": "CAP-GLY domain containing linker protein 3 (CLIP3 or CLIPR-59) is thought to function as an adaptor protein recruiting CYLD into the TNFR1 signaling to facilitate CYLD-mediated deubiquitination of RIPK1 in TNFalpha signaling (Fujikura D et al. 2012). CLIP3-assisted CYLD-mediated K63-deubiquitination of RIPK1 may promote caspase-8 activation to induce apoptosis by TNFalpha. The effects of CLIP3 (CLIPR-59) knockdown on apoptosis induction by TNFalpha were more effective in human cervical cancer HeLa cells than in human alveolar basal epithelial A549 cells or human fibrosarcoma HT1080 cells. These findings suggest that the role of CLIP3 on TNF-alpha-induced and RIP1-mediated pro-apoptotic signaling is dependent on cell type and context (Fujikura D et al. 2012). Further, low expression levels of CLIP3 (CLIPR-59) were detected in human glioblastoma multiforme (GBM) cells, such as U87MG, and high grade glioma biopsy samples (Ding Z et al. 2015). In malignant gliomas, a negative relationship between overexpression of speedy protein A (SPDYA or SPY1) and CLIP3 downregulation was associated with a highly aggressive phenotype of glioma (Ding Z et al. 2015; Kang H et al. 2021). In GBM cells, CLIP3 interacted with SPDYA, resulting in decreased association of CLIP3 with CYLD (Ding Z et al. 2015). This in turn led to reduced CYLD-mediated deubiquitination of RIPK1 suppressing TNF-\u03b1-induced cell death of glioma cells (Ding Z et al. 2015).", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q12933-1", "P98170-1", "Q13546-1", "Q13490-1", "Q13489-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-5357928", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-5357928" } ] }, { "id": "R-HSA-5696627", "name": "CYLD deubiquitinates K63polyUb-TRAF2,K63polyUb-TRAF6,K63polyUb-RIPK1,K63polyUb-IKBKG", "equation": "", "summary": "CYLD removes Lys63-linked ubiquitin chains, acting as a negative regulator of NF-kappa-B signaling (Trompouki et al. 2003). It deubiquitinates several NF-kappa-B regulators including TRAF2, TRAF6 (Kovalenko et al. 2003, Trompouki et al. 2003), IKBKG (NEMO) (Brummelkamp et al. 2003), and RIPK1 (Wright et al. 2007).", "cellular_location": "cytosol", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q12933-1", "Q13546-1", "Q9Y4K3-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-5696627", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-5696627" } ] }, { "id": "R-HSA-741411", "name": "CYLD deubiquitinates NEMO", "equation": "", "summary": "RIP2-induced ubiquitination of NEMO and consequent NFkappaB activation can be reversed in a dose-responsive manner by the deubiquitinase CYLD, suggesting that CYLD negatively regulates RIP2-induced NEMO ubiquitinylation.", "cellular_location": "cytosol", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "O43353-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-741411", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-741411" } ] }, { "id": "R-HSA-83582", "name": "TRADD:TRAF2:RIP1 complex dissociates from the TNF-:TNF-R1 complex.", "equation": "", "summary": "Once formed in context of the TNFR1 signaling complex the TRADD:TRAF2:RIPK1 complex may dissociate from the TNF:TNFR1 platform. With the recruitment of FADD and caspase-8 to the TRADD:TRAF2:RIPK1 complex the cell is pushed along the apoptotic pathway provided that the protective FLIP protein and TRAF2-associated BIRC (cIAPs) do not inhibit caspase-8 activation by RIPK1 and RIPK3-mediated activation of the necroptotic pathway.", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q96AX9-1", "Q12933-1", "Q14164-1", "Q9UHD2-1", "P98170-1", "Q13546-1", "Q13490-1", "Q13489-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-83582", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-83582" } ] }, { "id": "R-HSA-936390", "name": "CYLD mediated deubiquitination of DDX58 (RIG-I)", "equation": "", "summary": "CYLD is an ovarian tumor (OTU) domain-containing deubiquitinating enzyme (DUB) and has been identified as a negative regulator of DDX58 (RIG-I) mediated antiviral signaling. CYLD associates with the CARD domain of DDX58 and removes K63-linked ubiquitin from the DDX58 CARDs that are conjugated by the E3 ubiquitin ligase, TRIM25 and RNF135.", "cellular_location": "cytosol", "input_participant_list": [ "R-HSA-918191" ], "output_participant_list": [ "R-HSA-168917" ], "enzyme_list": [ "O95786-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-936390", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-936390" } ] }, { "id": "R-HSA-9793680", "name": "OPTN binds polyUb-RIPK1 within the TNFR1 complex", "equation": "", "summary": "Optineurin (OPTN, also called FIP2 and NRP), is a multifunctional adaptor protein that contains several domains including two coiled-coil domains, a leucine zipper (LZ) domain, an LC3-interacting region (LIR), a ubiquitin-binding domain (UBD), and a zinc finger (ZF) domain. OPTN has been implicated in regulating various signaling pathways including NF-kappa-B activation, TBK1-mediated type I interferon production, programmed cell death, and autophagy (reviewed in Markovinovic A et al. 2017; Slowicka K & van Loo G 2018; Toth RP & Atkin JD 2018). OPTN has been identified as a negative regulator of the tumor necrosis factor \u03b1 (TNF- \u03b1)-induced signaling pathway that targets several downstream components including receptor-interacting serine/threonine protein kinase 1 (RIPK1), caspase 8 (CASP8) and ubiquitin (Ub) carboxyl-terminal hydrolase CYLD (Zhu G et al. 2007; Nagabhushana A et al. 2011; Nakazawa S et al. 2016). TNF-\u03b1 binding to TNF receptor 1 (TNFR1) results in the sequential formation of several signaling complexes that trigger inflammatory responses and cell survival or cell death (Walczak H 2011; Ofengeim D & Yuan J 2013). The rapidly forming complex-I (TNFR1 signaling complex) is assembled at the receptor\u2019s cytoplasmic tail. The components of this complex, including RIPK1, are rapidly conjugated with Ub chains by various E3 ligases (Micheau O and Tschopp J 2003; Yuan J et al. 2019). The ubiquitination status (K63-, M1-linked or Ub-free) of RIPK1 determines cell fate downstream of the TNFR1 signaling complex. OPTN has been shown to associate with TNFR1 and RIPK1 by coimmunoprecipitation in a TNF-\u03b1-stimulated mouse embryonic fibroblasts (MEF) (Zhu G et al. 2007) and human epithelial carcinoma KB cell line (a subclone of HeLa cells) (Klingseisen L et al. 2012) suggesting that OPTN is recruited to the TNFR1 complex. In OPTN-deficient HeLa cells, IP assays show enhanced association of TNFR1 with ubiquitinated RIPK1 and two subunits of linear ubiquitin chain assembly complex (LUBAC), HOIL-1L and SHARPIN Nakazawa S et al. 2016). In contrast, overexpression of OPTN suppresses these associations within the TNFR1 signaling complex suggesting that OPTN binding downregulates the TNFR1 complex activity (Nakazawa S et al. 2016). Further, endogenous OPTN immunoprecipitates with polyubiquitinated RIPK1 in TNF-\u03b1-stimulated HeLa cells (Zhu G et al. 2007; Nakazawa S et al. 2016) and with anti-linear Ub antibody in TNF-\u03b1-stimulated HEK293T cells (Nakazawa S et al. 2016). In vitro pull-down assays show that maltose-binding protein (MBP)-fused OPTN (Nakazawa S et al. 2016) or glutathione S-transferase (GST)-fused OPTN (Gleason CE et al. 2011) can be copurified with M1- and K63-linked polyubiquitin, but not K48-linked polyubiquitin. Structural studies further confirm that OPTN functions through its ability to bind polyubiquitinated proteins showing that the UBD of OPTN preferentially recognizes linear M1-linked Ub chains (Nakazawa S et al. 2016; Li F et al. 2018). A surface plasmon resonance (SPR) analysis has revealed that OPTN binds to M1-linked Ub with the micromolar affinity (Nakazawa S et al. 2016). These findings suggest that the Ub-binding activity of OPTN downregulates the TNF-\u03b1-induced signaling pathway by targeting RIPK1 function within the TNFR1 signaling complex (Zhu G et al. 2007; Nakazawa S et al. 2016). However, the precise role of OPTN downstream of TNFR1 remains unclear. TNF-\u03b1 was found to induce OPTN expression in an NF-kappa-B-dependent manner in human lung adenocarcinoma A549 cells and HeLa cells (Sudhakar C et al. 2009). OPTN was shown to suppress activation of NF-kappa-B in TNF-\u03b1-stimulated HeLa cells (Sudhakar C et al. 2009; Nakazawa S et al. 2016) and human embryonic kidney 293T (HEK293T) cells (Munitic I et al. 2013). In addition, increased NF-kappa-B activity has been observed in OPTN-deficient mouse microglial BV2 cells, neuroblastoma Neuro2a cells and hybrid NSC-34 cells produced by fusing neuroblastoma and spinal-cord motor neurons (Akizuki M et al. 2013; Maruyama H et al. 2010; Cao LL et al. 2021). Further, the UBD-disrupting mutant OPTN D474N as well as amyotrophic lateral sclerosis (ALS)-associated mutation OPTN E478G, prevent binding of OPTN to polyUb (Gleason CE et al. 2011; Nakazawa S et al. 2016) and fail to suppress TNF-alpha-mediated NF-kappa-B activation in human and mouse cells (Zhu G et al. 2007; Sudhakar C et al. 2009; Maruyama H et al. 2010; Nakazawa S et al. 2016). Elevated expression of RIPK1 and downregulated expression of OPTN has been detected in the brains of Alzheimer's disease (AD) patients and the APP/PS1 mouse model of AD (Cao LL et al. 2021). Intracerebroventricular injection of adeno-associated virus (AAV) vector encoding OPTN to APP/PS1 mice reduces neuroinflammation in microglial cells and astrocytes via RIPK-dependent NF-kappa-B pathways (Cao LL et al. 2021). Although OPTN is believed to downregulate TNF-induced inflammation, in ex vivo studies in various primary cells, including bone marrow-derived macrophages, microglia and MEFs isolated from OPTN-deficient or OPTN-insufficient mice (carrying point mutation D477N or C-terminal truncation, which cannot bind polyubiquitin), OPTN had no effect on NF-kappa-B signaling, suggesting that the mechanism of OPTN-mediated regulation is indirect and/or depends on the NF-kappa-B-mediated upregulation of OPTN expression (Sudhakar C et al. 2009; Gleason CE et al. 2011; Munitic I et al. 2013; Slowicka K et al. 2016; Markovinovic A et al. 2018; reviewed in Slowicka K & van Loo G 2018; Prescott JA et al. 2021). Further, ectopic overexpression of OPTN results in decreased protein levels of RIPK1 without affecting the mRNA levels of RIPK1 in BV2 cells, suggesting that OPTN may regulate RIPK1 activity by enhancing RIPK1 degradation (Cao LL et al. 2021). Moreover, OPTN-deficient mice developed neuropathology resembling one aspect of ALS namely dysmyelination, and this neuropathy was associated with RIPK1-dependent induction of necroptosis in the spinal cord and was linked to decreased K48-ubiquitination and turnover of RIPK1 (Ito Y et al 2016). However, dysmyelination was not confirmed in a follow-up study (Dermentzaki G et al. 2019), which showed that unmanipulated Optn-deficient mice had no phenotype and that OPTN-deficiency had an effect on RIPK1 in vitro but not in vivo. The same study did not find any significant changes in RIPK1 levels in motor cortex samples from patients with ALS (Dermentzaki et al. 2019). Further research is needed to clarify the role of RIPK1-induced necroptosis in the pathogenesis of ALS (Ito Y et al 2016; Dermentzaki et al. 2019). Moreover, as an autophagy receptor, OPTN binds polyubiquitinated cargos through its UBD and delivers them to autophagosomes via LIR-mediated interaction with microtubule-associated protein 1A/1B (MAP1LC3 or LC3), a key initiator of autophagy. The autophagy receptor function of OPTN is enhanced by TANK-binding kinase 1 (TBK1)-mediated phosphorylation of OPTN (Li F et al. 2018). The N-terminal coiled-coil domain of OPTN directly interacts with the C-terminal domain of TBK1 to form a stable complex (Li F et al. 2018). Both OPTN and TBK1 have been shown to associate with the components of the TNFR1 signaling complex in the M1 (or K63)-linked polyUb-dependent manner to alter RIPK1 function (Zhu G et al. 2007; Nakazawa S et al. 2016; Lafont E et al. 2018; Xu D et al. 2018; Taft J et al. 2021). Notably, TBK1-mediated phosphorylation of OPTN on S473 has been shown to broaden the binding specificity of OPTN to other polyubiquitin chains including K48-linked polyUb (Li F et al. 2018). Similar to OPTN, TBK1 deficiency, which is embryonically lethal (Bonnard M et al. 2000), results in the TNF-\u03b1-stimulated hyperactivation of RIPK1 kinase activity leading to necroptosis in vitro and in vivo (Xu D et al. 2018; Taft J et al. 2021). Mutations of TBK1 that cannot bind OPTN have been linked to ALS and a related neurodegenerative diasease, frontotemporal dementia (FTD) (Freischmidt A et al. 2015; Cirulli ET et al. 2015). Further, TBK1 deficiency shows limited effect on TNF-induced gene expression in human A549 and mouse embryonic fibroblasts (MEF) (Lafont E et al. 2018). Thus, OPTN may participate in the crosstalk between TNF-\u03b1-induced cell death signaling and cytoprotective autophagy by targeting RIPK1-containing complexes for autophagic degradation. Besides RIPK1, OPTN may control the TNF-\u03b1-induced signaling by binding CYLD (Nagabhushana A et al. 2011) and pro-CASP8 (Nakazawa S et al. 2016). In addition, the OPTN:TBK1 axis regulates host immune responses downstream of pattern-recognition receptors (PRR) such as Toll-like receptor 3 (TLR3) or retinoic-acid-inducible protein 1 (RIG-I, also known as DDX58) (Meena NP et al. 2016; Pourcelot M et al. 2016; Markovinovic A et al. 2018; O\u2019Loughlin T et al. 2020). OPTN has been shown to translocate to Golgi-proximal foci in human retinal pigment epithelium (RPE) cells in response to TLR3 ligand (poly(I:C)) and DDX58 ligand (pppRNA) (O\u2019Loughlin T et al. 2020). OPTN-interacting proteins, including TBK1, CYLD and components of the LUBAC complex, have been identified in these foci, suggesting that OPTN inhibits the PRR-mediated innate immune response through sequestering key components of NF-kappa-B and TBK1:IRF3 signaling pathways in the perinuclear compartment (O\u2019Loughlin T et al. 2020).
This Reactome event shows OPTN association with ubiquitinated RIPK1 within the TNFR1 signaling complex.", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q12933-1", "P98170-1", "Q13546-1", "Q13490-1", "Q13489-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9793680", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9793680" } ] }, { "id": "R-HSA-9796379", "name": "CYLD:SPATA2:LUBAC binds TNFR1 signaling complex", "equation": "", "summary": "The N-terminal methionine-linked ubiquitin (Met1-Ub) chains, or linear Ub chains, are conjugated to target proteins by the linear ubiquitin chain assembly complex (LUBAC) composed of HOIL-1L, HOIP, and SHARPIN (Kirisako T et al. 2006; Walczak H et al. 2012). LUBAC and linear polyubiquitination has been implicated in the regulation of innate immunity and cell death (Tokunaga F et al. 2011; Gerlach B et al. 2011). LUBAC activity is regulated by deubiquitinases (DUBs) such as OTULIN and CYLD, which hydrolyze Ub chains to reverse the modification of target proteins (Takiuchi T et al. 2014). OTULIN and CYLD associate with HOIP, the catalytic subunit of LUBAC, to cleave Ub chains in a linkage-specific fashion. OTULIN exclusively hydrolyzes Met1-Ub, while CYLD disassembles both Met1-Ub and Lys63-Ub (Fiil BK et al. 2013; Harhaj EW and Dixit VM 2012; Hrdinka M et al. 2016; Sato Y 2022). The CYLD interaction with LUBAC is facilitated by spermatogenesis-associated protein 2 (SPATA2) (Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wagner SA et al. 2016). Genome-wide siRNA screen identified SPATA2 as a gene involved in mediating necroptosis (Hitomi J et al. 2008). Structural and biochemical studies revealed that the N-terminal PNGase/UBA or UBX (PUB) domain of SPATA2 interacts with the Ub-specific protease (USP) domain of CYLD, whereas the C-terminal PUB-interacting motif (PIM) of SPATA2 binds the PUB domain of HOIP (reviewed in Schlicher L et al. 2017; Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016). Under steady-state conditions, SPATA2 was found to bridge CYLD and LUBAC forming the trimeric CYLD:SPATA2:LUBAC complex (Wagner SA et al. 2016; Elliott PR et al. 2016; reviewed in Schlicher L et al. 2017). In TNF\u03b1-stimulated human and mouse cells, CYLD:SPATA2:LUBAC is rapidly recruited into the TNFR1 signaling complex (Wagner SA et al. 2016; Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wei R et al. 2017; reviewed in Schlicher L et al. 2017). Knockdown of SPATA2 selectively abolished CYLD interaction with the TNFR1 complex, without abolishing binding of LUBAC components (Wagner SA et al. 2016). Within the TNFR1 signaling complex, SPATA2 promotes the deubiquitinase activity of CYLD resulting in CYLD-mediated removal of Met1- and K63-linked polyubiquitin chains from specific substrates such as RIPK1 (Wei R et al. 2017). SPATA2 deficiency increased the Met1-linked ubiquitination of RIPK1, which resulted in inhibition of RIPK1 kinase activity and RIPK1-mediated cell death in mouse cells (Wei R et al. 2017). SPATA2 deficiency promoted the production of proinflammatory cytokines in TNF\u03b1-treated mice in vivo (Wei R et al. 2017). Further, SPATA2 deficiency protected against TNF-induced cell death in human and mouse cells (Wagner SA et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wei R et al. 2017). These data suggest that SPATA2 regulates inflammatory signaling and cell death program via linking CYLD and LUBAC to limit LUBAC-mediated Met1-Ub.
This Reactome event shows the recruitment of the CYLD:SPATA2:LUBAC complex to the TNFR1 signaling complex. ; ;", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q12933-1", "P98170-1", "Q13546-1", "Q13490-1", "Q13489-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9796379", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9796379" } ] }, { "id": "R-HSA-9796691", "name": "SPATA2 links CYLD to LUBAC", "equation": "", "summary": "Genome-wide siRNA screening identified spermatogenesis-associated protein 2 (SPATA2 ) as a gene involved in mediating necroptosis (Hitomi J et al. 2008). Under steady-state conditions, SPATA2 was shown to interact with deubiquitinating enzyme CYLD and HOIP, the catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC) (Wagner SA et al. 2016; Elliott PR et al. 2016; reviewed in Schlicher L et al. 2017). Structural and biochemical studies revealed that the N-terminal PNGase/UBA or UBX (PUB) domain of SPATA2 interacts with the USP domain of CYLD, whereas the C-terminal PUB-interacting motif (PIM) of SPATA2 binds the PUB domain of HOIP (reviewed in Schlicher L et al. 2017; Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016). In TNF\u03b1-stimulated human and mouse cells, CYLD:SPATA2:LUBAC is rapidly recruited into the TNFR1 signaling complex (Wagner SA et al. 2016; Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wei R et al. 2017; reviewed in Schlicher L et al. 2017). Knockdown of SPATA2 selectively abolished CYLD interaction with the TNFR1 complex, without abolishing binding of LUBAC components (Wagner SA et al. 2016). Within the TNFR1 signaling complex, SPATA2 promotes the deubiquitinase activity of CYLD resulting in CYLD-mediated removal of Met1- and K63-linked polyubiquitin chains from specific substrates such as RIPK1 (Wei R et al. 2017). SPATA2 deficiency increased the Met1-linked ubiquitination of RIPK1, which resulted in inhibition of RIPK1 kinase activity and RIPK1-mediated cell death in mouse cells (Wei R et al. 2017). SPATA2 deficiency promoted the production of proinflammatory cytokines in TNF\u03b1-treated mice in vivo (Wei R et al. 2017). Further, SPATA2 deficiency protected against TNF-induced cell death in human and mouse cells (Wagner SA et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wei R et al. 2017). These data suggest that SPATA2 regulates inflammatory signaling and cell death program via linking CYLD and LUBAC to limit LUBAC-mediated Met1-Ub.
The Reactome event shows the formation of the CYLD:SPATA2:LUBAC complex. The stoichiometry of the trimeric CYLD:SPATA2:LUBAC complex is shown as 2:2:2 (Elliott PR et al. 2016). ;", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9796691", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9796691" } ] }, { "id": "R-HSA-9815507", "name": "MIB2 ubiquitinates CYLD at K338, K530", "equation": "", "summary": "The E3 ubiquitin (Ub) ligase activity of mind bomb 2 (MIB2) protein controls the tumor necrosis factor alpha (TNF\u03b1)-induced pathway by targeting several downstream components including receptor-interacting serine/threonine protein kinase 1 (RIPK1), Ub carboxyl-terminal hydrolase CYLD and FLICE-like inhibitory protein long (FLIP(L) encoded by the CFLAR gene) (Feltham R et al. 2018; Uematsu A et al. 2019; Nakabayashi O et al. 2021). 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 TNF\u03b1-induced pro-survival responses while promoting RIPK1-mediated cell death (reviewed in Ju E et al. 2022). As a deubiquitinating enzyme, CYLD removes K63-linked and Met1-linked polyUb chains from RIPK1 and other substrates downstream of TNFR1, thus enhancing cytotoxic potential of RIPK1 (Uematsu A et al. 2019). Upon co-expression of tagged CYLD and MIB2 in human embryonic kidney 293T (HEK293T) cells, MIB2 directly binds to and catalyzes K48-linked polyubiquitination of CYLD thus targeting CYLD for proteasomal degradation (Rajan N et al. 2014; Uematsu A et al. 2019). Immunoaffinity purification using antibodies that detect ubiquitin remnant motif (K-Epsilon-GG) on modified Lys residues coupled with quantitative mass spectrometry analysis revealed that MIB2 ubiquitinates CYLD at K338 and K530 upon co-expression in HEK293T cells. Mutagenesis analysis confirmed that MIB2-dependent ubiquitination of CYLD occurs at K338 and K530 (Uematsu A et al. 2019). MIB2-mediated degradation of CYLD is thought to prevent CYLD-mediated deubiquitination of RIPK1 suppressing RIPK1-dependent cell death (Uematsu A et al. 2019). Ub-conjugating Enzyme E2D 2 (UBE2D2) facilitated MIB2-mediated ubiquitination of CYLD in in vitro assay. Besides CYLD, MIB2 directly binds and ubiquitinates RIPK1 within the TNF\u03b1:TNFR1 signaling complex I inhibiting TNF\u03b1-induced cell death (Feltham R et al. 2018). These data suggest that the E3 ligase activity of MIB2 suppresses the TNF-induced cell death by attaching K48-linked polyUb chains to CYLD (Feltham R et al. 2018; Uematsu A et al. 2019; Nakabayashi O et al. 2021)
This Reactome event describes MIB2-mediated K48-linked ubiquitination of CYLD in the presence of UBE2D2.", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96AX9-1", "P51668-1", "P62837-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9815507", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9815507" } ] }, { "id": "R-HSA-9815510", "name": "MIB2 binds CYLD", "equation": "", "summary": "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 (TNF\u03b1)-induced pathway. Ligation of TNF\u03b1 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 TNF\u03b1 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 TNF\u03b1-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 TNF\u03b1: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 TNF\u03b1-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 TNF\u03b1-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).", "cellular_location": "cytosol", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96AX9-1", "P62837-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9815510", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9815510" } ] }, { "id": "R-HSA-9817362", "name": "SPATA2:CYLD-bound LUBAC ubiquitinates RIPK1 at K627 within the TNFR1 signaling complex", "equation": "", "summary": "TNF-\u03b1 ligation to TNF receptor 1 (TNFR1) induces the formation of the TNFR1 signaling complex composed of TNFR1, TRADD (TNFR1-associated death domain), TRAF2 (TNF receptor associated factor-2), RIPK1 (receptor-interacting serin/threonine protein kinase 1), and E3 ubiquitin ligases BIRC2, BIRC3 (cIAP1/2, cellular inhibitor of apoptosis) and LUBAC (linear ubiquitin chain assembly complex) (Micheau O and Tschopp J 2003). The conjugation of K63-linked ubiquitin (Ub) chains by BIRC2/3 to RIPK1 allows further recruitment and activation of the TAK1 (also known as MAP3K7) complex and I\u03baB kinase (IKK) complex which in turn drives activation of NF-kappa-B (Ea CK et al. 2006; Haas et al. 2009). The catalytic activity of LUBAC, composed of HOIL-1L, HOIP, and SHARPIN, specifically generates the N-terminal methionine-linked ubiquitin (Met1-Ub) chains (also known as linear Ub chains) (Kirisako T et al. 2006; Walczak H et al. 2012; Rittinger K & Ikeda F 2017; Fuseya Y & Iwai K 2021). UBE2L3 functions as E2-conjugating enzyme for LUBAC (Lewis MJ et al. 2015; Fu B et al. 2014). LUBAC was shown to catalyze Met1-linked ubiquitination of RIPK1 at K627 upon co-expression of Flag-RIPK1 with Myc-tagged components of LUBAC (HOIP, HOIL1, and SHARPIN) in human embryonic kidney 293T (HEK293T) cells (Tu H et al. 2021). Linear ubiquitination of mouse Ripk1 on K612 (K627 in human) inhibited TNF-\u03b1\u2013induced apoptosis and necroptosis in mouse embryonic fibroblast (MEF) cells. Mutagenesis analysis further confirmed the importance of the K627 (K612 in mouse) ubiquitination site of RIPK1 in the TNFR1 signaling pathway (Li X et al. 2020). Further, LUBAC is known to ubiquitinate IKBKG (NEMO), a regulatory component of the IKK complex. LUBAC-mediated IKBKG ubiquitination enhanced IKBKG interaction with the TNFR1 signaling complex and stabilized this protein complex to promote activation of NF-kappa-B (Haas TL et al. 2009). Importantly, deletion of the LUBAC component SHARPIN in mice or mutation of HOIL-1 in humans, leads to hyperinflammatory phenotypes, indicating key roles of LUBAC and linear Ub chains in the response to infection and inflammation (Gerlach B et al. 2011; Ikeda F et al. 2011; Tokunaga F et al. 2011; Boisson B et al. 2012). The data suggest that LUBAC-mediated linear ubiquitylation of RIPK1 limits TNF-\u03b1\u2013induced cell death. LUBAC activity is regulated by deubiquitinases (DUBs) such as OTULIN and CYLD, which hydrolyze Ub chains to reverse the modification of target proteins (Takiuchi T et al. 2014). OTULIN and CYLD associate with HOIP, the catalytic subunit of LUBAC, to cleave Ub chains in a linkage-specific fashion. OTULIN exclusively hydrolyzes Met1-Ub, while CYLD disassembles both Met1-Ub and Lys63-Ub (Fiil BK et al. 2013; Harhaj EW and Dixit VM 2012; Hrdinka M et al. 2016; Sato Y 2022). The CYLD interaction with LUBAC is facilitated by spermatogenesis-associated protein 2 (SPATA2) (Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wagner SA et al. 2016). The CYLD:SPATA2:LUBAC complex is rapidly recruited into the TNFR1 signaling complex to regulate ubiquitination/deubiquitination downstream of TNFR1 (Wagner SA et al. 2016; Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wei R et al. 2017; reviewed in Schlicher L et al. 2017).
This Reactome event describes LUBAC-mediated Met1-linked polyubiquitination (M1polyUb) of RIPK1 at K627 within the TNFR1 signaling complex.", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q12933-1", "P98170-1", "Q13546-1", "P68036-1", "Q13490-1", "Q13489-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9817362", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9817362" } ] }, { "id": "R-HSA-9817397", "name": "TBK1, IKBKE phosphorylate RIPK1 at T189", "equation": "", "summary": "Binding of TNF\u03b1 to TNF receptor 1 (TNFR1) induces either cell survival or cell death through the sequential formation of several signaling complexes, namely complex-I, IIa and IIb (Micheau O and Tschopp J 2003; Walczak H 2011). The dynamic assembly of these complexes is tightly regulated by proteolysis, ubiquitination, deubiquitination and phosphorylation of receptor-interacting serine/threonine protein kinase 1 (RIPK1) and other components of the TNF\u03b1 signaling pathway. The rapidly forming complex-I (the TNFR1 signaling complex) is assembled at the receptor\u2019s cytoplasmic tail and consists of TNFR1, TNFR1-associated death domain (TRADD), TNF receptor associated factor-2 (TRAF2) and RIPK1 (Micheau O and Tschopp J 2003). The activated TNFR1 signaling complex (complex-I) recruits several E3 ubiquitin (Ub) ligases, such as cIAP1/2 cellular inhibitor of apoptosis (BIRC2, BIRC3), mind bomb 2 (MIB2) or linear ubiquitin chain assembly complex (LUBAC) (Micheau O and Tschopp J 2003; Feltham R et al. 2018; Yuan J et al. 2019). K63-linked ubiquitination by BIRC2, BIRC3 and MIB2 as well as LUBAC-mediated Met1-linked linear ubiquitination of RIPK1 and other complex components stabilize the membrane-bound pro-survival TNFR1 signaling complex, while suppressing the formation of the cytosolic death-inducing complex IIa (TRADD:TRAF2:RIPK1:FADD:CASP8) and IIb (RIPK1:RIPK3:MLKL). The catalytic activity of LUBAC also enables recruitment of TRAF-associated NF-\u03baB activator (TANK) binding kinase 1 (TBK1) and inhibitor-kappa-B kinase (IKK) epsilon (IKK\u03b5 or IKBKE) to the TNFR1 signaling complex (Lafont E et al. 2018; Xu D et al. 2018). LUBAC is composed of the HOIP, HOIL-1L, and SHARPIN subunits. HOIP deficiency strongly diminished recruitment of TBK1 or IKBKE to the complex-I in human cervical carcinoma epithelial HeLa cells and lung carcinoma epithelial A549 cells (Lafont E et al. 2018). Western blot analysis revealed that TBK1 and IKBKE are recruited to and strongly phosphorylated within the TNFR1 complex in various TNF\u03b1-stimulated human cell lines including A549, keratinocyte HaCaT, monocytic U937 and THP-1 cells (Lafont E et al. 2018). Similarly, TBK1 was found to associate with components of the complex-I in the LUBAC-dependent manner in mouse cells (Xu D et al. 2018). IKBKE, but not TBK1, was detected within the TNFR1 complex in human telomerase reverse transcriptase (TERT)-immortalized dermal fibroblasts derived from patients with TBK1 deficiency as a result of loss-of-function mutation at W619* (Taft J et al. 2021). The enzymatic activity of TBK1/IKBKE is initiated by phosphorylation at S172 located in the T loop of the TBK1 and IKK\u03b5 kinase domains (Shimada T et al. 1999; Kishore N et al. 2002; Gu L et al. 2013). Activated TBK1 and IKBKE are known to trigger phosphorylation of interferon regulatory factor 3 (IRF3) and IRF7 and subsequent expression of type I interferons (IFNs; IFN-\u03b1/\u03b2) downstream of pattern recognition receptors, such as Toll-like receptors 3 and 4 (TLR3, TLR4), cGAS/STING and RIG-I-like receptors (Fitzgerald KA et al. 2003; Fang R et al. 2017). While TBK1 or IKBKE showed limited effect on TNF-induced gene expression in human A549 and mouse embryonic fibroblasts (MEF) (Lafont E et al. 2018), both kinases prevented TNF\u03b1-induced cell death by suppressing RIPK1 activation in human and mouse cells (Lafont E et al. 2018; Xu D et al. 2018; Taft J et al. 2021). Mechanistically, TBK1 and/or IKBKE directly phosphorylate RIPK1 within the TNFR1 signaling complex to prevent RIPK1 kinase-dependent cell death signaling downstream of TNFR1 (Lafont E et al. 2018; Xu D et al. 2018). Mass spectrometry analysis suggests that human RIPK1 is phosphorylated by TBK1 at T189 (Xu D et al. 2018). Using a phospho-specific antibody, T189 of RIPK1 was detected as a phosphorylation site upon incubation with TBK1 in in vitro kinase assay and upon co-expression of tagged proteins in human embryonic kidney HEK293T cells. This phosphorylation was inhibited by TBK1/IKBKE inhibitor MRT67307 (Xu D et al. 2018). Phosphorylation of endogenous RIPK1 at T189 was detected in TNF-stimulated Jurkat cells (Xu D et al. 2018). Others reported that TBK1 or IKBKE phosphorylate RIPK1 at multiple residues (Lafont E et al. 2018). Further, TBK1 or IKBKE deficiency enhanced TNF-induced autophosphorylation of RIPK1, association of RIPK3 with caspase-8:FADD and levels of phosphorylated MLKL in human A549 and HeLa cells (Lafont E et al. 2018). Similar findings were reported for mouse cells (Lafont E et al. 2018; Xu D et al. 2018). In vivo studies showed that the embryonic lethality of Tbk1\u2212/\u2212 mice was caused by TNF\u03b1-stimulated hyperactivation of RIPK1 kinase activity (Xu D et al. 2018). Moreover, chronic systemic autoinflammation in patients carrying homozygous loss-of-function mutations in TBK1 (W619*, R440* and Y212D) is associated with enhanced TNF\u03b1-induced RIPK1-dependent cell death (Taft J et al. 2021). In addition, TBK1-mediated phosphorylation of CYLD may also control the TNFR1 signaling pathway by regulating the ubiquitination status of RIPK1 (Xu X et al. 2020; Taft J et al. 2021). These data suggest that TBK1 and IKBKE downregulate RIPK1 auto-phosphorylation within the complex-I, thereby preventing TNF\u03b1- induced RIPK1-kinase-activity-dependent cell death.
This Reactome event describes TBK1/IKBKE-mediated phosphorylation of RIPK1 at T189 within the TNFR1 signaling complex.", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q12933-1", "Q14164-1", "Q9UHD2-1", "P98170-1", "Q13546-1", "Q13490-1", "Q13489-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9817397", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9817397" } ] }, { "id": "R-HSA-9817400", "name": "CYLD hydrolyses K63polyUb on RIPK1 within the TNFR1 complex", "equation": "", "summary": "Binding of TNF\u03b1 to TNF receptor 1 (TNFR1) induces the sequential formation of several signaling complexes, namely complex I and complex IIa/b (Walczak H 2011; Yuan J et al. 2019). These complexes support either cell survival (complex I) or cell death (complex II). 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 TNF\u03b1 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 polyubiquitin-bound RIPK1 serves as a scaffold in the TNFR1 signaling complex (complex I) to recruit TAK1 kinase complex and I-kappa-B kinase (IKK) complex 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 leads to the activation of RIPK1 kinase activity, which promotes RIPK1 kinase-dependent cell death (reviewed in Ju E et al. 2022). Several deubiquitinating enzymes, such as tumor necrosis factor alpha-induced protein 3 (TNFAIP3, known as Zn finger protein A20) or ubiquitin (Ub) carboxyl-terminal hydrolase CYLD, have been implicated in the regulation of RIPK1 activity. CYLD disassembles both Met1- and Lys63-polyUb chains (Fiil BK et al. 2013; Harhaj EW and Dixit VM 2012; Hrdinka M et al. 2016; Sato Y 2022). Upon TNF\u03b1 stimulation, CYLD is recruited to the TNFR1 signaling complex along with the linear ubiquitin chain assembly complex (LUBAC). Interaction of CYLD with LUBAC is facilitated by spermatogenesis-associated protein 2 (SPATA2) (Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wagner SA et al. 2016). The CYLD:SPATA2:LUBAC complex is rapidly recruited into the TNFR1 signaling complex to regulate ubiquitination/deubiquitination downstream of TNFR1 (Wagner SA et al. 2016; Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wei R et al. 2017; reviewed in Schlicher L et al. 2017).
This Reactome event describes CYLD-mediated cleavage of K63-linked polyubiquitin chains on RIPK1 within the TNFR1 signaling complex. The deubiquitinase activity of CYLD attenuates TNF\u03b1-induced NF-kappa-B and MAPK signaling, but promotes cell death.
",
"cellular_location": "cytosol, plasma membrane",
"input_participant_list": [],
"output_participant_list": [],
"enzyme_list": [
"Q96EP0-1",
"Q9BYM8-1",
"Q12933-1",
"P98170-1",
"Q13546-1",
"Q13490-1",
"Q13489-1",
"Q9NQC7-1"
],
"evidence": [
{
"id": "R-HSA-9817400",
"database": "Reactome",
"url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9817400"
}
]
},
{
"id": "R-HSA-9817411",
"name": "TBK1, IKBKE binds Met1-polyUb within the TNFR1 complex",
"equation": "",
"summary": "The catalytic activity of linear ubiquitin chain assembly complex (LUBAC) enables recruitment of TRAF-associated NF-\u03baB activator (TANK) binding kinase 1 (TBK1) and inhibitor-kappa-B kinase (IKK) epsilon (IKK\u03b5 or IKBKE) to the TNFR1 signaling complex, also known as complex-I (Lafont E et al. 2018; Xu D et al. 2018). LUBAC, composed of the HOIP, HOIL-1L, and SHARPIN subunits, generates linear Met1-linked polyubiquitin chains. HOIP deficiency strongly diminished recruitment of TBK1 or IKBKE to the complex-I in human epithelial cervical carcinoma HeLa cells and lung carcinoma A549 cells (Lafont E et al. 2018). Western blot analysis revealed that TBK1 and IKBKE are recruited to and strongly phosphorylated within the TNFR1 complex in various TNF\u03b1-stimulated human cell lines including A549, keratinocyte HaCaT, monocytic U937 and THP-1 cells (Lafont E et al. 2018). Similarly, TBK1 was found to associate with components of the complex-I in the LUBAC-dependent manner in mouse cells (Xu D et al. 2018). IKBKE, but not TBK1, was detected within the TNFR1 complex in human telomerase reverse transcriptase (TERT)-immortalized dermal fibroblasts derived from patients with TBK1 deficiency as a result of loss-of-function mutation at W619* (Taft J et al. 2021). The enzymatic activity of TBK1/IKBKE is initiated by phosphorylation at S172 located in the T loop of the TBK1 and IKK\u03b5 kinase domains (Shimada T et al. 1999; Kishore N et al. 2002; Gu L et al. 2013). Activated TBK1 and IKBKE are known to trigger phosphorylation of interferon regulatory factor 3 (IRF3) and IRF7 and subsequent expression of type I interferons (IFNs; IFN-\u03b1/\u03b2) downstream of pattern recognition receptors, such as Toll-like receptors 3 and 4 (TLR3, TLR4), cGAS/STING and RIG-I-like receptors (Fitzgerald KA et al. 2003; Fang R et al. 2017). While TBK1 or IKBKE showed limited effect on TNF\u03b1-induced gene expression in human A549 and mouse embryonic fibroblasts (MEF) (Lafont E et al. 2018), both kinases prevented TNF\u03b1-induced cell death by suppressing RIPK1 activation in human and mouse cells (Lafont E et al. 2018; Xu D et al. 2018; Taft J et al. 2021). TBK1 and/or IKBKE directly phosphorylate RIPK1 within the TNFR1 signaling complex to prevent RIPK1 kinase-dependent cell death downstream of TNFR1 (Lafont E et al. 2018; Xu D et al. 2018). TBK1 or IKBKE deficiency enhanced TNF-induced autophosphorylation of RIPK1, association of RIPK3 with caspase-8:FADD and phosphorylation of MLKL in human A549 and HeLa cells (Lafont E et al. 2018). Similar findings were reported for mouse cells (Lafont E et al. 2018; Xu D et al. 2018). In vivo studies showed that the embryonic lethality of Tbk1\u2212/\u2212 mice was caused by TNF\u03b1-stimulated hyperactivation of RIPK1 kinase activity (Xu D et al. 2018). Furthermore, chronic systemic autoinflammation in patients carrying homozygous loss-of-function mutations in TBK1 (W619*, R440* and Y212D) is associated with the enhanced TNF\u03b1-induced RIPK1-dependent cell death (Taft J et al. 2021). In addition, TBK1-mediated phosphorylation of CYLD may also control the TNFR1 signaling pathway by regulating the ubiquitination status of RIPK1 (Xu X et al. 2020; Taft J et al. 2021). These data suggest that TBK1 and IKBKE downregulate RIPK1 auto-phosphorylation within the complex-I, thereby preventing TNF\u03b1- induced RIPK1-kinase-activity-dependent cell death.
This Reactome event describes TBK1/IKBKE recruitment to and activation within the TNFR1 signaling complex.", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q12933-1", "Q14164-1", "Q9UHD2-1", "P98170-1", "Q13546-1", "Q13490-1", "Q13489-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9817411", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9817411" } ] }, { "id": "R-HSA-9818789", "name": "CHUK, IKBKB phosphorylate RIPK1 at S25", "equation": "", "summary": "Binding of TNF\u03b1 to TNF receptor 1 (TNFR1) induces the sequential formation of several signaling complexes, namely complex I and complex IIa/b (Walczak H 2011; Yuan J et al. 2019). These complexes support either cell survival (complex I) or cell death (complex II). The dynamic 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 TNF\u03b1 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 polyubiquitin-bound RIPK1 serves as a scaffold in the TNFR1 signaling complex (complex I) to recruit TAK1 kinase complex and I-kappa-B kinase (IKK) complex 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 leads to the activation of RIPK1 kinase activity, which promotes autophosphorylation of RIPK1 and RIPK1 kinase-dependent cell death (reviewed in Ju E et al. 2022). The kinase activity of RIPK1 is regulated by phosphorylation (Lafont E et al. 2018; Dondelinger Y et al. 2019; reviewed in Delanghe T et al. 2020; Ju E et al. 2022). The components of I-kappa-B kinase complex, namely inhibitor of nuclear factor kappa B subunit \u03b1 (IKK\u03b1 or CHUK) and subunit \u03b2 (IKK\u03b2 or IKBKB), can directly phosphorylate RIPK1 within the membrane-bound TNFR1 signaling complex (complex I) (Dondelinger Y et al. 2015, 2019). IKK phosphorylates S25 in the kinase domain of RIPK1 to block ATP binding, preventing RIPK1 kinase activation and cell death (Dondelinger Y et al. 2019). Expression of phospho-mimetic S25D RIPK1 mutant prevented TNF-induced cell death in RIPK1-null human Jurkat cells and mouse embryonic fibroblasts (MEF). Autophosphorylation of RIPK1 on S166 and activation of RIPK1 kinase were inhibited in mouse bone marrow-derived macrophages (BMDMs) and MEFs isolated from mice carrying S25D RIPK1 mutant (Dondelinger Y et al. 2019; Puylaert P et al. 2022). Inhibition of IKK activity by Yersinia infection or by SHARPIN gene mutations results in defective phosphorylation of RIPK1 at S25 by IKK\u03b1/\u03b2 (Dondelinger Y et al. 2019). Restoring S25 phosphorylation with S25D RIPK1 prevented TNF-induced RIPK1 kinase-dependent cell death in Yersinia-infected BMDMs. Further, mimicking S25 phosphorylation impaired host immune response to Yersinia infection in vivo in bone marrow chimeric mice, suggesting that RIPK1 kinase-dependent cell death contributes to immune defense in response to pathogens. Similarly, S25D RIPK1 suppressed TNF-induced cell death in mouse dermal fibroblasts (MDFs) isolated from SHARPIN-deficient mice. These data suggest that IKK\u03b1/\u03b2-mediated phosphorylation of RIPK1 at S25 downregulates RIPK1 auto-phosphorylation downstream of TNFR1, thereby preventing TNF\u03b1-induced RIPK1 kinase-dependent cell death (Dondelinger Y et al. 2019).
This Reactome event describes IKK mediated phosphorylation of RIPK1 at S25 within the TNFR1 signaling complex.; ; ;", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q12933-1", "O15111-1", "P98170-1", "Q13546-1", "O14920-1", "Q13490-1", "Q13489-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9818789", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9818789" } ] }, { "id": "R-HSA-9818975", "name": "CYLD hydrolyses M1polyUb on RIPK1 within the TNFR1 complex", "equation": "", "summary": "Binding of TNF\u03b1 to TNF receptor 1 (TNFR1) induces the sequential formation of several signaling complexes, namely complex I and complex IIa/b (Walczak H 2011; Yuan J et al. 2019). These complexes support either cell survival (complex I) or cell death (complex II). 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 TNF\u03b1 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 polyubiquitin-bound RIPK1 serves as a scaffold in the TNFR1 signaling complex (complex I) to recruit TAK1 kinase complex and I-kappa-B kinase (IKK) complex 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 leads to the activation of RIPK1 kinase activity, which promotes RIPK1 kinase-dependent cell death (reviewed in Ju E et al. 2022). Several deubiquitinating enzymes, such as tumor necrosis factor alpha-induced protein 3 (TNFAIP3, known as Zn finger protein A20) or ubiquitin (Ub) carboxyl-terminal hydrolase CYLD, have been implicated in the regulation of RIPK1 activity. CYLD disassembles both Met1- and Lys63-polyUb chains (Fiil BK et al. 2013; Harhaj EW and Dixit VM 2012; Hrdinka M et al. 2016; Sato Y 2022). Upon TNF\u03b1 stimulation, CYLD is recruited to the TNFR1 signaling complex along with the linear ubiquitin chain assembly complex (LUBAC). Interaction of CYLD with LUBAC is facilitated by spermatogenesis-associated protein 2 (SPATA2) (Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wagner SA et al. 2016). The CYLD:SPATA2:LUBAC complex is rapidly recruited into the TNFR1 signaling complex to regulate ubiquitination/deubiquitination downstream of TNFR1 (Wagner SA et al. 2016; Elliott PR et al. 2016; Kupka S et al. 2016; Schlicher L et al. 2016; Wei R et al. 2017; reviewed in Schlicher L et al. 2017).
This Reactome event describes CYLD-mediated cleavage of Met1-linked polyubiquitin chains on RIPK1 within the TNFR1 signaling complex. The deubiquitinase activity of CYLD attenuates TNF\u03b1-induced NF-kappa-B and MAPK signaling, but promotes cell death.
",
"cellular_location": "cytosol, plasma membrane",
"input_participant_list": [],
"output_participant_list": [],
"enzyme_list": [
"Q96EP0-1",
"Q9BYM8-1",
"Q12933-1",
"P98170-1",
"Q13546-1",
"Q13490-1",
"Q13489-1",
"Q9NQC7-1"
],
"evidence": [
{
"id": "R-HSA-9818975",
"database": "Reactome",
"url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9818975"
}
]
},
{
"id": "R-HSA-9824874",
"name": "OPTN recruits CYLD to the TNFR1 complex",
"equation": "",
"summary": "Optineurin (OPTN) is a multifunctional, ubiquitin-binding adaptor protein which exhibits a high homology with IKBKG (NEMO, the regulatory subunit of the IkB kinase complex). OPTN has been implicated in regulating of various signaling pathways including NF-kappa-B activation, TBK1-mediated type I interferon production, programmed cell death, and autophagy (reviewed in Markovinovic A et al. 2017; Slowicka K & van Loo G 2018; Toth RP & Atkin JD 2018). In human and mouse cells, OPTN was identified as a negative regulator of the tumor necrosis factor \u03b1 (TNF-\u03b1)-induced signaling pathway by targeting several downstream components including receptor-interacting serine/threonine protein kinase 1 (RIPK1), caspase 8 (CASP8), and ubiquitin (Ub) carboxyl-terminal hydrolase CYLD (Zhu G et al. 2007; Nagabhushana A et al. 2011; Nakazawa S et al. 2016). Direct association of OPTN with TNFR1 and RIPK1 was detected in TNF-\u03b1-stimulated human epithelial carcinoma KB cells and HeLa cells by coimmunoprecipitation suggesting that OPTN is recruited to the TNFR1 complex (Zhu G et al. 2007; Klingseisen L et al. 2012; Nakazawa S et al. 2016). Structural and biochemical studies showed that OPTN functions through its ability to bind ubiquitinated proteins such as RIPK1, and the UBAN domain of OPTN preferentially recognizes linear polyUb chains (Gleason CE et al. 2011; Nakazawa S et al. 2016; Li F et al. 2018). Although the highest affinity is seen for M1-linked polyUb chains, TBK1-mediated phosphorylation of OPTN on S473 has been shown to broaden its specificity to include K63- and K48-linked polyUb chains (Li F et al. 2018). The Ub-binding activity of OPTN is thought to downregulate the TNF-\u03b1 mediated NF-kappa-B activation (Zhu G et al. 2007; Maruyama H et al. 2010; Nakazawa S et al. 2016) by suppressing a scaffold function of RIPK1 within the TNFR1 signaling complex (Zhu G et al. 2007; Nakazawa S et al. 2016). In addition, OPTN interacts with deubiquitinase CYLD, which acts as a negative regulator of the TNFR1-induced gene expression by hydrolyzing K63-, M1-linked polyUb chains of RIPK1. CoIP assay detected interaction of OPTN with CYLD upon co-expression of tagged proteins in HeLa cells (Nagabhushana A et al. 2011). Defective binding of the glaucoma-associated OPTN H486R variant to CYLD prevented CYLD-mediated inhibition of TNF\u03b1-induced NF-kappa-B activity upon co-expression of OPTN and CYLD in HeLa cells. A catalytically inactive CYLD variant co-immunoprecipitated polyUb-RIPK1 along with OPTN in HeLa cells. Further, OPTN knockdown prevented RIPK1 deubiquitination by overexpressed CYLD in TNF-\u03b1-induced HeLa cells (Nagabhushana A et al. 2011). The data suggest that OPTN directly interacts with CYLD and RIPK1 to mediate deubiquitination of RIPK1 by CYLD within the TNFR1 signaling complex thus suppressing the NF-kappa-B signaling pathway. However, the OPTN regulatory function on NF-kappa-B signaling observed in vitro has not been confirmed in vivo suggesting that the mechanism of OPTN-mediated regulation is indirect and/or depends on the NF-kappa-B-mediated upregulation of OPTN (Sudhakar C et al. 2009; Gleason CE et al. 2011; Munitic I et al. 2013; Slowicka K et al. 2016; Markovinovic A et al. 2018).
Similar to mutations in the OPTN gene, CYLD mutations have been linked to the development of neurodegenerative diseases (Dobson-Stone C et al. 2020; Gu X et al. 2021; Xiao X et al. 2022). For example, the missense CYLD mutation M719V was identified in a family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis (FTD-ALS) (Dobson-Stone C et al. 2020). When overexpressed in HEK293 cells, CYLD M719V has been shown to increase deubiquitinating activity of CYLD, leading to enhanced inhibition of the NF-kappa-B pathway, suggesting that this gain-of-function mechanism of action may contribute to the development of FTD-ALS (Dobson-Stone C et al. 2020).
This Reactome event shows CYLD association with OPTN bound to the TNFR1 signaling complex.", "cellular_location": "cytosol, plasma membrane", "input_participant_list": [], "output_participant_list": [], "enzyme_list": [ "Q96EP0-1", "Q9BYM8-1", "Q12933-1", "P98170-1", "Q13546-1", "Q13490-1", "Q13489-1", "Q9NQC7-1" ], "evidence": [ { "id": "R-HSA-9824874", "database": "Reactome", "url": "https://www.reactome.org/PathwayBrowser/#R-HSA-9824874" } ] } ], "reaction_participants": [ { "participant_id": "R-HSA-168917", "participant_name": "DDX58 [cytosol]", "crossref": [ { "id": "O95786", "database": "UniProt", "url": "" }, { "id": "5E3H", "database": "PDB", "url": "" }, { "id": "5F98", "database": "PDB", "url": "" }, { "id": "4P4H", "database": "PDB", "url": "" }, { "id": "4ON9", "database": "PDB", "url": "" }, { "id": "5F9H", "database": "PDB", "url": "" }, { "id": "2920", "database": "Guide to Pharmacology - Targets", "url": "" }, { "id": "NP_055129.2", "database": "RefSeq", "url": "" }, { 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"uniprot_ac": "O15111", "gene_name": "CHUK", "activity": [ { "ec_number": "2.7.11.10", "activity": "ATP + L-seryl-[I-kappa-B protein] = ADP + H(+) + O-phospho-L-seryl-[I-kappa-B protein].", "evidence": [ { "id": "O15111", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/O15111" } ] }, { "ec_number": "2.7.11.10", "activity": "", "evidence": [ { "id": "O15111", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/O15111" } ] } ] }, { "uniprot_ac": "O43318", "gene_name": "MAP3K7", "activity": [ { "ec_number": "2.7.11.25", "activity": "ATP + L-seryl-[protein] = ADP + H(+) + O-phospho-L-seryl-[protein].", "evidence": [ { "id": "O43318", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/O43318" } ] }, { "ec_number": "2.7.11.25", "activity": "", "evidence": [ { "id": "O43318", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/O43318" } ] } ] }, { "uniprot_ac": "O43353", "gene_name": "RIPK2", "activity": [ { "ec_number": "2.7.10.2", 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}, { "ec_number": "3.4.19.12", "activity": "", "evidence": [ { "id": "O75604", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/O75604" } ] } ] }, { "uniprot_ac": "O95786", "gene_name": "RIGI", "activity": [ { "ec_number": "3.6.4.13", "activity": "ATP + H2O = ADP + H(+) + phosphate.", "evidence": [ { "id": "O95786", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/O95786" } ] }, { "ec_number": "3.6.4.13", "activity": "", "evidence": [ { "id": "O95786", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/O95786" } ] } ] }, { "uniprot_ac": "P21580", "gene_name": "TNFAIP3", "activity": [ { "ec_number": "3.4.19.12", "activity": "Thiol-dependent hydrolysis of ester, thioester, amide, peptide and isopeptide bonds formed by the C-terminal Gly of ubiquitin (a 76-residue protein attached to proteins as an intracellular targeting signal).", "evidence": [ { "id": "P21580", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/P21580" } ] }, { "ec_number": "3.4.19.12", "activity": "", "evidence": [ { "id": "P21580", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/P21580" } ] }, { "ec_number": "2.3.2.-", "activity": "", "evidence": [ { "id": "P21580", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/P21580" } ] } ] }, { "uniprot_ac": "P51668", "gene_name": "UBE2D1", "activity": [ { "ec_number": "2.3.2.24", "activity": "S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [acceptor protein]-L-lysine = [E1 ubiquitin-activating enzyme]-L-cysteine + N(6)-monoubiquitinyl-[acceptor protein]-L-lysine.", "evidence": [ { "id": "P51668", "database": "UniProtKB", "url": "https://www.uniprot.org/uniprot/P51668" } ] }, { "ec_number": "2.3.2.23", "activity": "S-ubiquitinyl-[E1 ubiquitin-activating enzyme]-L-cysteine + [E2 ubiquitin-conjugating enzyme]-L-cysteine = [E1 ubiquitin-activating enzyme]-L-cysteine + S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine.", "evidence": [ { "id": 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1 } ], "sort_fields": [ "glytoucan_ac", "type", "subtype", "site_category", "site_seq", "relation", "comment" ] }, { "table_id": "glycosylation_predicted", "table_stats": [ { "field": "total", "count": 0 }, { "field": "total_sites", "count": 0 }, { "field": "n_linked_glycans", "count": 0 }, { "field": "n_linked_glycan_sites", "count": 0 }, { "field": "o_linked_glycans", "count": 0 }, { "field": "o_linked_glycan_sites", "count": 0 } ], "sort_fields": [] }, { "table_id": "glycosylation_automatic_literature_mining", "table_stats": [ { "field": "total", "count": 0 }, { "field": "total_sites", "count": 0 }, { "field": "n_linked_glycans", "count": 0 }, { "field": "n_linked_glycan_sites", "count": 0 }, { "field": "o_linked_glycans", "count": 0 }, { "field": "o_linked_glycan_sites", "count": 0 } ], "sort_fields": [] }, { "table_id": "phosphorylation", "table_stats": [ { "field": "total", "count": 29 }, { "field": "total_sites", "count": 12 } ], "sort_fields": [ "start_pos", "end_pos", "kinase_uniprot_canonical_ac", "kinase_gene_name", "residue", "comment", "site_lbl" ] }, { "table_id": "snv", "table_stats": [ { "field": "total", "count": 1 }, { "field": "total_sites", "count": 1 } ], "sort_fields": [ "start_pos", "end_pos", "subjects_tested", "subjects_positive", "frequency", "sequence_org", "sequence_mut", "comment", "chr_id", "chr_pos", "ref_nt", "alt_nt", "minor_allelic_frequency", "site_lbl" ] }, { "table_id": "snv_disease", "table_stats": [ { "field": "total", "count": 1 }, { "field": "total_sites", "count": 1 } ], "sort_fields": [ "start_pos", "end_pos", "subjects_tested", "subjects_positive", "frequency", "sequence_org", "sequence_mut", "comment", "chr_id", "chr_pos", "ref_nt", "alt_nt", "minor_allelic_frequency", "site_lbl" ] }, { "table_id": "snv_non_disease", "table_stats": [ { "field": "total", "count": 0 }, { "field": "total_sites", "count": 0 } ], "sort_fields": [] }, { "table_id": "publication", "table_stats": [ { "field": "total", "count": 202 } ], "sort_fields": [ "title", "journal", "date", "authors" ] } ] }