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LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-ĸB to the nucleus

ABSTRACT: Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling, however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here we show that the NF-ĸB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-ĸB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-ĸB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-kB activation, both serving as a signaling platform, as well as a transport mode for activated NF-kB to the nuclear.

SUBMITTER: Prof. Konstanze, F Winklhofer

PROVIDER: S-SCDT-10_15252-EMBJ_2022112006 | biostudies-other |

REPOSITORIES: biostudies-other

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LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Project description:Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

| S-EPMC9753471 | biostudies-literature

Reciprocal interplay between OTULIN-LUBAC determines genotoxic and inflammatory NF-κB signal responses.

Project description:Targeting nuclear factor-kappa B (NF-κB) represents a highly viable strategy against chemoresistance in cancers as well as cell death. Ubiquitination, including linear ubiquitination mediated by the linear ubiquitin chain assembly complex (LUBAC), is emerging as a crucial mechanism of overactivated NF-κB signaling. Ovarian tumor family deubiquitinase OTULIN is the only linear linkage-specific deubiquitinase; however, the molecular mechanisms of how it counteracts LUBAC-mediated NF-κB activation have been largely unknown. Here, we identify Lys64/66 of OTULIN for linear ubiquitination facilitated in a LUBAC-dependent manner as a necessary event required for OTULIN-LUBAC interaction under unstressed conditions, which becomes deubiquitinated by OTULIN itself in response to genotoxic stress. Furthermore, this self-deubiquitination of OTULIN occurs intermolecularly, mediated by OTULIN dimerization, resulting in the subsequent dissociation of OTULIN from the LUBAC complex and NF-κB overactivation. Oxidative stress induces OTULIN dimerization via cysteine-mediated covalent disulfide bonds. Our study reveals that the status of the physical interaction between OTULIN and LUBAC is a crucial determining factor for the genotoxic NF-κB signaling, as measured by cell survival and proliferation, while OTULIN loss of function resulting from its dimerization and deubiquitination leads to a dissociation of OTULIN from the LUBAC complex. Of note, similar molecular mechanisms apply to the inflammatory NF-κB signaling in response to tumor necrosis factor α. Hence, a fuller understanding of the detailed molecular mechanisms underlying the disruption of the OTULIN-LUBAC interaction will be instrumental for developing future therapeutic strategies against cancer chemoresistance and necroptotic processes pertinent to numerous human diseases.

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Termination of NF-kappaB activity through a gammaherpesvirus protein that assembles an EC5S ubiquitin-ligase.

Project description:Host colonisation by lymphotropic gammaherpesviruses depends critically on the expansion of viral genomes in germinal centre (GC) B cells. Yet, host and virus molecular mechanisms involved in driving such proliferation remain largely unknown. Here, we show that the ORF73 protein encoded by the murid herpesvirus-4 (MuHV-4) inhibits host nuclear factor-kappa B (NF-kappaB) transcriptional activity through poly-ubiquitination and subsequent proteasomal-dependent nuclear degradation of the NF-kappaB family member p65/RelA. The mechanism involves the assembly of an ElonginC/Cullin5/SOCS (suppressors of cytokine signalling)-like complex, mediated by an unconventional viral SOCS-box motif present in ORF73. Functional deletion of this SOCS-box motif ablated NF-kappaB inhibitory effect of ORF73, suppressed MuHV-4 expansion in GC B cells and prevented MuHV-4 persistent infection in mice. These findings demonstrate that viral inhibition of NF-kappaB activity in latently infected GC centroblasts is critical for the establishment of a gammaherpesvirus persistent infection, underscoring the physiological importance of proteasomal degradation of RelA/NF-kappaB as a regulatory mechanism of this signalling pathway.

| S-EPMC2664658 | biostudies-literature

Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform.

Project description:The dynamics and coordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required the earliest, followed by auto-phosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps, whereas ULK1 and ULK2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way, suggesting multiple initiation events. Targeted ubiquitinated mitochondria are cradled by endoplasmic reticulum (ER) strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands, providing platforms for formation of the mitophagosomes.

| S-EPMC6739445 | biostudies-literature

The linear ubiquitin chain assembly complex (LUBAC) generates heterotypic ubiquitin chains.

Project description:The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RING-in-between-RING (RBR) domains, a coordinated ubiquitin relay mechanism between the HOIL-1-interacting protein (HOIP) and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.

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The fructose-2,6-bisphosphatase TIGAR suppresses NF-?B signaling by directly inhibiting the linear ubiquitin assembly complex LUBAC.

Project description:The systems integration of whole-body metabolism and immune signaling are central homeostatic mechanisms necessary for maintenance of normal physiology, and dysregulation of these processes leads to a variety of chronic disorders. However, the intracellular mechanisms responsible for cell-autonomous cross-talk between the inflammatory signaling pathways and metabolic flux have remained enigmatic. In this study, we discovered that the fructose-2,6-bisphosphatase TIGAR (Tp53-induced glycolysis and apoptosis regulator) critically regulates NF-?B activation. We found that TIGAR potently inhibits NF-?B-dependent gene expression by suppressing the upstream activation of IKK? phosphorylation and kinase activation. This inhibition occurred through a direct binding competition between NEMO and TIGAR for association with the linear ubiquitination assembly complex (LUBAC). This competition prevented linear ubiquitination of NEMO, which is required for activation of IKK? and other downstream targets. Furthermore, a TIGAR phosphatase activity-deficient mutant was equally effective as WT TIGAR in inhibiting NEMO linear ubiquitination, IKK? phosphorylation/activation, and NF-?B signaling, indicating that TIGAR's effect on NF-?B signaling is due to its interaction with LUBAC. Physiologically, TIGAR knockout mice displayed enhanced adipose tissue NF-?B signaling, whereas adipocyte-specific overexpression of TIGAR suppressed adipose tissue NF-?B signaling. Together, these results demonstrate that TIGAR has a nonenzymatic molecular function that modulates the NF-?B signaling pathway by directly inhibiting the E3 ligase activity of LUBAC.

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