OPTN binds polyUb-RIPK1 within the TNFR1 complex

Stable Identifier
R-HSA-9793680
Type
Reaction [binding]
Species
Homo sapiens
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ReviewStatus
5/5
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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 α (TNF- α)-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-α 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’s 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-α-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-α-stimulated HeLa cells (Zhu G et al. 2007; Nakazawa S et al. 2016) and with anti-linear Ub antibody in TNF-α-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-α-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-α 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-α-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-α-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-α-induced cell death signaling and cytoprotective autophagy by targeting RIPK1-containing complexes for autophagic degradation. Besides RIPK1, OPTN may control the TNF-α-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’Loughlin 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’Loughlin 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’Loughlin T et al. 2020).

This Reactome event shows OPTN association with ubiquitinated RIPK1 within the TNFR1 signaling complex.

Literature References
PubMed ID Title Journal Year
27552911 Linear ubiquitination is involved in the pathogenesis of optineurin-associated amyotrophic lateral sclerosis

Sawasaki, T, Nureki, O, Takeyoshi, I, Takahashi, H, Hatada, I, Iwai, K, Tokunaga, F, Oikawa, D, Ishii, R, Takeda, H, Ito, H, Ayaki, T, Kawakami, H, Nakazawa, S, Kamei, K, Ishitani, R

Nat Commun 2016
17702576 Optineurin negatively regulates TNFalpha- induced NF-kappaB activation by competing with NEMO for ubiquitinated RIP

Wu, CJ, Zhao, Y, Zhu, G, Ashwell, JD

Curr Biol 2007
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