ABSTRACT
The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.
Subject(s)
Immunologic Deficiency Syndromes , Nerve Tissue Proteins , Ubiquitins , Humans , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Female , Male , NF-kappa B/metabolism , Ubiquitin-Protein Ligases/genetics , Inflammation/immunology , Inflammation/genetics , B-Lymphocytes/immunology , Loss of Function Mutation , Fibroblasts/metabolism , Fibroblasts/immunology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Animals , Mice , AllelesABSTRACT
The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.
Subject(s)
Carrier Proteins/metabolism , Cell Death , Embryonic Development , Hematopoiesis , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Caspase 8/genetics , Caspase 8/metabolism , Cell Death/genetics , Embryo Loss/genetics , Embryonic Development/genetics , Endothelial Cells/cytology , Female , Hematopoiesis/genetics , Mice , Mice, Inbred C57BL , Protein Domains , Protein Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/geneticsABSTRACT
Ubiquitination (also known as ubiquitylation) is a post-translational modification that creates versatility in cell signalling and regulates a multitude of cellular processes. Its versatility lies in the capacity to form eight different inter-ubiquitin linkages through the seven lysine residues of ubiquitin and through its N-terminal methionine (M1). The latter, referred to as linear or M1 linkage, is created by the linear ubiquitin chain assembly complex (LUBAC), the only E3 ligase known to date that is capable of forming linear ubiquitin chains de novo Linear ubiquitin chains are crucial modulators of innate and adaptive immune responses, and act by regulating inflammatory and cell death signalling. In this Cell Science at a Glance article and the accompanying poster, we review the current knowledge on the role of LUBAC and linear ubiquitination in immune signalling and human physiology. We specifically focus on the role for LUBAC in signalling that is induced by the cytokine tumour necrosis factor (TNF) and its role in inflammation, gene activation and cell death. Furthermore, we highlight the roles of deubiquitinases (DUBs) that cleave M1 linkages and add an additional layer in the control of LUBAC-mediated immune signalling.
Subject(s)
Signal Transduction , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Ubiquitination , Animals , Cell Death , Humans , Inflammation/metabolism , Inflammation/pathology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.
Subject(s)
Immunity, Innate , Inflammation/immunology , Nod2 Signaling Adaptor Protein/metabolism , X-Linked Inhibitor of Apoptosis Protein/genetics , Animals , Female , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nod2 Signaling Adaptor Protein/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Signal Transduction , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
Ubiquitination is a post-translational modification that creates versatility in cell signalling, in part because eight biochemically different inter-ubiquitin linkages can be formed through the seven internal lysine residues of ubiquitin or its amino-terminal methionine. The latter, referred to as linear or M1 linkage, is created by the linear ubiquitin chain assembly complex (LUBAC). Previously, K63 linkages were thought to be exclusively responsible for ubiquitin-mediated nondegradative functions. It now emerges, however, that M1 ubiquitination is crucial in various pathways, and that generation of a physiological signalling output requires cooperation between different ubiquitin linkage types. Here, we review the currently known functions of LUBAC and M1 ubiquitination, discuss promising future research directions into their functions, and how this may reveal novel therapeutic opportunities for diseases with perturbed linear ubiquitination.
Subject(s)
Multiprotein Complexes/metabolism , Signal Transduction , Ubiquitin/metabolism , Ubiquitination , Animals , Humans , Lysine/metabolism , Methionine/metabolism , Models, BiologicalABSTRACT
Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1ß was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.
Subject(s)
Immunity/immunology , Inflammation/metabolism , Signal Transduction , Ubiquitination , Animals , CD40 Ligand/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line , Humans , I-kappa B Kinase/metabolism , Inflammation/pathology , Inflammation/prevention & control , Interleukin-1beta/metabolism , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , NF-kappa B/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Phenotype , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptors, Tumor Necrosis Factor/deficiency , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Skin/cytology , Skin/immunology , Skin/metabolism , Skin/pathology , Transcription Factors , Tumor Necrosis Factor-alpha/deficiency , Tumor Necrosis Factor-alpha/genetics , Ubiquitin/chemistry , Ubiquitin/metabolism , Ubiquitin-Protein Ligase Complexes/chemistry , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-kappaB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBAC's enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC.
Subject(s)
Gene Expression Regulation , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/physiology , Ubiquitin/metabolism , Animals , Apoptosis , Cell Line , GTPase-Activating Proteins/genetics , HeLa Cells , Humans , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/physiology , Intracellular Signaling Peptides and Proteins/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , NF-kappa B/metabolism , Signal Transduction , TNF Receptor-Associated Death Domain Protein/genetics , TNF Receptor-Associated Factor 2/genetics , TNF Receptor-Associated Factor 2/physiology , U937 Cells , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/physiologyABSTRACT
The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double-stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.
Subject(s)
RNA Viruses , Ubiquitin-Protein Ligases , Ubiquitination , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Signal Transduction , DEAD Box Protein 58/genetics , RNA Viruses/metabolismABSTRACT
Autoinflammatory diseases are a heterogenous group of disorders defined by fever and systemic inflammation suggesting involvement of genes regulating innate immune responses. Patients with homozygous loss-of-function variants in the OTU-deubiquitinase OTULIN suffer from neonatal-onset OTULIN-related autoinflammatory syndrome (ORAS) characterized by fever, panniculitis, diarrhea, and arthritis. Here, we describe an atypical form of ORAS with distinct clinical manifestation of the disease caused by two new compound heterozygous variants (c.258G>A (p.M86I)/c.500G>C (p.W167S)) in the OTULIN gene in a 7-year-old affected by a life-threatening autoinflammatory episode with sterile abscess formation. On the molecular level, we find binding of OTULIN to linear ubiquitin to be compromised by both variants; however, protein stability and catalytic activity is most affected by OTULIN variant p.W167S. These molecular changes together lead to increased levels of linear ubiquitin linkages in patient-derived cells triggering the disease. Our data indicate that the spectrum of ORAS patients is more diverse than previously thought and, thus, supposedly asymptomatic individuals might also be affected. Based on our results, we propose to subdivide the ORAS into classical and atypical entities.
Subject(s)
Endopeptidases , Hereditary Autoinflammatory Diseases/genetics , Ubiquitin , Child , Endopeptidases/genetics , Humans , Infant, Newborn , Inflammation/genetics , Ubiquitin/metabolismABSTRACT
Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies.
Subject(s)
ADAM17 Protein/antagonists & inhibitors , Endothelial Cells/metabolism , Necroptosis , Neoplasms/etiology , Neoplasms/pathology , Animals , Antineoplastic Agents/pharmacology , Biomarkers , Biomarkers, Tumor , Cell Communication , Cell Death , Disease Susceptibility/immunology , Humans , Necroptosis/genetics , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Seeding , Neoplasms/metabolism , Neoplasms/therapy , Proteolysis , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The linear-ubiquitin chain assembly complex (LUBAC) modulates signalling via various immune receptors. In tumour necrosis factor (TNF) signalling, linear (also known as M1) ubiquitin enables full gene activation and prevents cell death. However, the mechanisms underlying cell death prevention remain ill-defined. Here, we show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNF receptor 1 signalling complex (TNFR1-SC). While exerting only limited effects on TNF-induced gene activation, TBK1 and IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1 and IKKε phosphorylate the kinase RIPK1 in the TNFR1-SC, thereby preventing RIPK1-dependent cell death. This activity is essential in vivo, as it prevents TNF-induced lethal shock. Strikingly, NEMO (also known as IKKγ), which mostly, but not exclusively, binds the TNFR1-SC via M1 ubiquitin, mediates the recruitment of the adaptors TANK and NAP1 (also known as AZI2). TANK is constitutively associated with both TBK1 and IKKε, while NAP1 is associated with TBK1. We discovered a previously unrecognized cell death checkpoint that is mediated by TBK1 and IKKε, and uncovered an essential survival function for NEMO, whereby it enables the recruitment and activation of these non-canonical IKKs to prevent TNF-induced cell death.
Subject(s)
I-kappa B Kinase/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , A549 Cells , Animals , Cell Death/drug effects , Cells, Cultured , HeLa Cells , Humans , Mice, Knockout , Phosphorylation/drug effects , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction/drug effects , Ubiquitination/drug effectsABSTRACT
The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone.
Subject(s)
Carrier Proteins/metabolism , Dermatitis/metabolism , Fas Ligand Protein/pharmacology , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Ubiquitin-Protein Ligases/metabolism , Animals , Animals, Newborn , Carrier Proteins/genetics , Cell Death/drug effects , Cell Death/genetics , Cells, Cultured , Dermatitis/genetics , Intracellular Signaling Peptides and Proteins , Keratinocytes/cytology , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Skin/drug effects , Skin/metabolism , Skin/pathology , Ubiquitin-Protein Ligases/geneticsSubject(s)
Apoptosis/genetics , Multiprotein Complexes/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction , Transcriptional Activation , Tumor Necrosis Factor-alpha/metabolism , Ubiquitin/metabolism , Animals , Humans , Mice , Models, Biological , Polyubiquitin/metabolism , Protein BindingABSTRACT
The linear ubiquitin chain assembly complex (LUBAC), consisting of SHANK-associated RH-domain-interacting protein (SHARPIN), heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), and HOIL-1-interacting protein (HOIP), is a critical regulator of inflammation and immunity. This is highlighted by the fact that patients with perturbed linear ubiquitination caused by mutations in the Hoip or Hoil-1 genes, resulting in knockouts of these proteins, may simultaneously suffer from immunodeficiency and autoinflammation. TLR3 plays a crucial, albeit controversial, role in viral infection and tissue damage. We identify a pivotal role of LUBAC in TLR3 signaling and discover a functional interaction between LUBAC components and TLR3 as crucial for immunity to influenza A virus infection. On the biochemical level, we identify LUBAC components as interacting with the TLR3-signaling complex (SC), thereby enabling TLR3-mediated gene activation. Absence of LUBAC components increases formation of a previously unrecognized TLR3-induced death-inducing SC, leading to enhanced cell death. Intriguingly, excessive TLR3-mediated cell death, induced by double-stranded RNA present in the skin of SHARPIN-deficient chronic proliferative dermatitis mice (cpdm), is a major contributor to their autoinflammatory skin phenotype, as genetic coablation of Tlr3 substantially ameliorated cpdm dermatitis. Thus, LUBAC components control TLR3-mediated innate immunity, thereby preventing development of immunodeficiency and autoinflammation.
Subject(s)
Immunologic Deficiency Syndromes/metabolism , Inflammation/pathology , Nerve Tissue Proteins/metabolism , Signal Transduction , Toll-Like Receptor 3/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Death/drug effects , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Dermatitis/pathology , Gene Silencing/drug effects , Host-Pathogen Interactions/immunology , Humans , Inflammation/immunology , Influenza A virus/drug effects , Influenza A virus/physiology , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice , Poly I-C/pharmacology , Signal Transduction/drug effects , Toll-Like Receptor 3/deficiencyABSTRACT
The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3+ regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation.
Subject(s)
Cell Differentiation/physiology , Homeostasis/physiology , T-Lymphocytes/physiology , Thymus Gland/cytology , Ubiquitin/metabolism , Animals , Base Sequence , Cells, Cultured , Computational Biology , Gene Expression Regulation/physiology , Genotype , Mice , Protein Multimerization , Protein Processing, Post-Translational , RNA/genetics , Sequence Analysis, RNA , T-Lymphocytes/classification , Ubiquitin/chemistry , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Ubiquitination and deubiquitination are crucial for assembly and disassembly of signaling complexes. LUBAC-generated linear (M1) ubiquitin is important for signaling via various immune receptors. We show here that the deubiquitinases CYLD and A20, but not OTULIN, are recruited to the TNFR1- and NOD2-associated signaling complexes (TNF-RSC and NOD2-SC), at which they cooperate to limit gene activation. Whereas CYLD recruitment depends on its interaction with LUBAC, but not on LUBAC's M1-chain-forming capacity, A20 recruitment requires this activity. Intriguingly, CYLD and A20 exert opposing effects on M1 chain stability in the TNF-RSC and NOD2-SC. While CYLD cleaves M1 chains, and thereby sensitizes cells to TNF-induced death, A20 binding to them prevents their removal and, consequently, inhibits cell death. Thus, CYLD and A20 cooperatively restrict gene activation and regulate cell death via their respective activities on M1 chains. Hence, the interplay between LUBAC, M1-ubiquitin, CYLD, and A20 is central for physiological signaling through innate immune receptors.
Subject(s)
Cell Death/physiology , Transcriptional Activation/physiology , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin/metabolism , Ubiquitination/physiology , Cell Line , DNA-Binding Proteins/metabolism , Deubiquitinating Enzyme CYLD , Humans , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Signal Transduction/physiology , Transduction, Genetic , Tumor Necrosis Factor alpha-Induced Protein 3 , Tumor Suppressor Proteins/metabolismABSTRACT
Linear ubiquitination is crucial for innate and adaptive immunity. The linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP, and SHARPIN, is the only known ubiquitin ligase that generates linear ubiquitin linkages. HOIP is the catalytically active LUBAC component. Here, we show that both constitutive and Tie2-Cre-driven HOIP deletion lead to aberrant endothelial cell death, resulting in defective vascularization and embryonic lethality at midgestation. Ablation of tumor necrosis factor receptor 1 (TNFR1) prevents cell death, vascularization defects, and death at midgestation. HOIP-deficient cells are more sensitive to death induction by both tumor necrosis factor (TNF) and lymphotoxin-α (LT-α), and aberrant complex-II formation is responsible for sensitization to TNFR1-mediated cell death in the absence of HOIP. Finally, we show that HOIP's catalytic activity is necessary for preventing TNF-induced cell death. Hence, LUBAC and its linear-ubiquitin-forming activity are required for maintaining vascular integrity during embryogenesis by preventing TNFR1-mediated endothelial cell death.
Subject(s)
Embryo Loss/metabolism , Endothelial Cells/cytology , Receptors, Tumor Necrosis Factor, Type I/metabolism , Ubiquitin-Protein Ligases/deficiency , Animals , Apoptosis/physiology , Cell Death/physiology , Embryo Loss/genetics , Embryo, Mammalian , Endothelial Cells/metabolism , Female , Lymphotoxin-alpha/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Receptors, Tumor Necrosis Factor, Type I/genetics , Tumor Necrosis Factor-alpha/pharmacology , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Yolk Sac/blood supplyABSTRACT
The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis/physiology , Carrier Proteins/physiology , Caspases/physiology , Membrane Glycoproteins/physiology , Tumor Necrosis Factor-alpha/physiology , fas Receptor/physiology , Animals , Apoptosis Regulatory Proteins , B-Lymphocytes/metabolism , CHO Cells , Caspase 10 , Caspase 8 , Caspase 9 , Caspases/metabolism , Cricetinae , Cricetulus , Enzyme Induction , Fas-Associated Death Domain Protein , Humans , Jurkat Cells/metabolism , Lymphocyte Activation , Macromolecular Substances , Protein Isoforms/physiology , Protein Structure, Tertiary , Protein Transport , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction , T-Lymphocytes/metabolism , TNF-Related Apoptosis-Inducing LigandABSTRACT
The Newcastle disease virus (NDV) has antineoplastic and immunostimulatory properties, and it is currently clinically tested in anticancer therapy. However, the tumoricidal mechanisms of NDV tumor therapy are not fully understood. The results presented here demonstrate that NDV-stimulated human monocytes (Mphi) kill various human tumor cell lines and that this tumoricidal activity is mediated by TRAIL. In contrast to soluble TRAIL-R2-Fc, soluble CD95-Fc and TNF-R2-Fc showed only minimal blocking of the antitumor effect. TRAIL expression is induced on human Mphi after stimulation with NDV and UV-inactivated NDV. These results show that TRAIL induction on human Mphi after NDV stimulation is independent from viral replication and that TRAIL mediates the tumoricidal activity of NDV-stimulated human Mphi.