Linear Ubiquitin Chain Assembly Complex Components Research Articles

LUBAC is required for RIG-I sensing of RNA viruses.

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.

TSG101 Physically Interacts with Linear Ubiquitin Chain Assembly Complex (LUBAC) and Upregulates the TNFα-Induced NF-κB Activation.

Linear ubiquitin chain assembly complex (LUBAC) is a ubiquitin E3 ligase complex composed of HOIP, HOIL-1L, and SHARPIN that catalyzes the formation of linear/M1- linked ubiquitin chain. It has been shown to play a pivotal role in the nuclear factor (NF)-κB signaling induced by proinflammatory stimuli. Here, we found that tumor susceptibility gene (TSG101) physically interacts with HOIP, a catalytic component of LUBAC, and potentiates LUBAC activity. Depletion of TSG101 expression by RNA interference decreased TNFα-induced linear ubiquitination and the formation of TNFα receptor 1 signaling complex (TNFRSC). Furthermore, TSG101 facilitated the TNFα-induced stimulation of the NF-κB pathway. Thus, we suggest that TSG101 functions as a positive modulator of HOIP that mediates TNFα-induced NF-κB signaling pathway.

Abstract 4980: Design and functional characterization of a first-in-class irreversible inhibitor of HOIL-1-interacting protein (HOIP) with selective antitumor activity against B-cell non-Hodgkin lymphoma

Abstract Activating single-nucleotide polymorphisms of HOIL-1-interacting protein (HOIP), the catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC), have recently been shown to promote myeloid differentiation primary response 88 (MYD88)-mediated B-cell lymphomagenesis. To assess the relevance of targeting HOIP in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) with MYD88 mutation, we used systems biology to build a mathematical model aimed at evaluating the impact of HOIP depletion on DLBCL. From this model, an AI-mediated query demonstrated that HOIP blockade was associated with the suppression of three main pathophysiological motifs in malignant B cells, i.e., cell growth and proliferation, apoptosis evasion and deregulated metabolism. We then undertook a computational study that consisted of a combinatorial substitution of several α-β unsaturated moieties to be used as covalent binding warheads to the catalytic cysteine residue of HOIP. The resulting chemical library was used on subsequent molecular docking to assess the best HOIP binding candidates. Out of the four candidates synthesized from this library, we isolated compound A (Cpd A), a covalent irreversible inhibitor of HOIP with a pyrido[2,3-d]pyrimidine core, which exerted selective antitumor activity in a panel of ABCL-DLBCL cell lines (mean IC50 at 48hours: 90.7± 13.09 µM) while sparing normal B cells. Immunoprecipitation studies demonstrated that Cpd A was able to modulate the interaction between HOIP and the LUBAC component, SHARPIN, leading to the blockade of NF-κB signaling and to the downregulation of several downstream effectors, including CCL3, IL6 and IRF4. Specificity of Cpd A towards HOIP was confirmed by a drug affinity responsive target stability (DARTS) assay based on the immuno-detection of persistent HOIP peptides after Cpd A-mediated blockade of enzymatic proteolysis, and thereafter in a CRISPR-engineered HOIP-knockout ABC-DLBCL model. Finally, efficacy of the compound was confirmed in vivo in a chicken embryo chorioallantoic membrane (CAM)-derived model of ABCL-DLBCL subjected to a twice weekly dosing with Cpd A, in which the compound achieved a 25% tumor growth inhibition, associated with a 60.5% and 89% reduction in brain and bone marrow infiltration by lymphoma cells, respectively. Altogether, our results confirm that HOIP represents a promising therapeutic target for ABC-DLBCL with activating MYD88 mutation, and that a pyrido[2,3-d]pyrimidine derivative can successfully and specifically block HOIP, resulting in NF-κB disruption and selective antitumor activity in this aggressive subtype of B-cell lymphoma. Citation Format: Ana Maria Montagut, Marc Antoni Armengol Cubillos, Gema Gorjón-de-Pablo, Judith Farrés, Laia Huguet Garcia, Núria Profitós-Pelejà, Juliana Carvalho Santos, Marcelo Lima Ribeiro, Miranda Fernández-Serrano, Roger Estrada Tejedor, José I. Borrell, Gaël Roué. Design and functional characterization of a first-in-class irreversible inhibitor of HOIL-1-interacting protein (HOIP) with selective antitumor activity against B-cell non-Hodgkin lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4980.

RNF31 promotes tumorigenesis via inhibiting RIPK1 kinase-dependent apoptosis.

It is well established that interferon (IFN) and tumor necrosis factor (TNF) could synergistically promote antitumor toxicity and avoid resistance of antigen-negative tumors during cancer immunotherapy. The linear ubiquitin chain assembly complex (LUBAC) has been widely known to regulate receptor-interacting protein kinase-1(RIPK1) kinase activity and TNF-mediated cell death during inflammation and embryogenesis. However, whether LUBAC and RIPK1 kinase activity in tumor microenvironment could regulate antitumor immunity are still not very clear. Here, we demonstrated a cancer cell-intrinsic role of LUBAC complex in tumor microenvironment to promote tumorigenesis. Lacking LUBAC component RNF31 in B16 melanoma cells but not immune cells including macrophages or dendritic cells greatly impaired tumor growth by increasing intratumoral CD8+ T cells infiltration. Mechanistically, we found that tumor cells without RNF31 shown severe apoptosis-mediated cell death caused by TNFα/IFNγ in the tumor microenvironment. Most importantly, we found that RNF31 could limit RIPK1 kinase activity and further prevent tumor cell death in a transcription-independent manner, suggesting a crucial role of RIPK1 kinase activity in tumorigenesis. Together, our results demonstrate an essential role of RNF31 and RIPK1 kinase activity in tumorigenesis and imply that RNF31 inhibition could be harnessed to enhance antitumor toxicity during tumor immunotherapy.

HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation.

HOIL‐1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase‐inactive HOIL‐1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL‐1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL‐1 monoubiquitylates glycogen and α1:4‐linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester‐linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100‐fold by the interaction of Met1‐linked or Lys63‐linked ubiquitin oligomers with the RBR domain of HOIL‐1. HOIL‐1 also transferred pre‐formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL‐1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL‐1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.

Deficiency of the E3 Ligase Hoip Impairs Adult Hematopoiesis and Myeloid Leukemia

Ubiquitination is a post-translational modification that plays important roles in the regulation of various cellular processes. The linear ubiquitin chain assembly complex (LUBAC) is the only E3 ubiquitin ligase, which can specifically generate linear ubiquitin chains. LUBAC is composed of three subunits: catalytic Hoip (Rnf31), Hoil-1l (RBCK1), and Sharpin. LUBAC regulates canonical NF-κB signaling pathway and apoptosis. Several studies have shown that LUBAC is required for fetal hematopoiesis and tumorigenesis in several solid cancers, but the role of Hoip, which is the catalytic component of the LUBAC, in adult hematopoiesis and myeloid leukemia is unclear.To address whether Hoip is required for adult hematopoiesis, we used tamoxifen induced Hoip deleted mice (Hoip fl/fl; Rosa26-CreERT). Deletion of Hoip reduced the numbers of almost all cell fractions in bone marrow and peripheral blood, except for long-term hematopoietic stem cell (LT-HSC) fraction (CD34 - Flk2 - Lineage - c-Kit + Sca-1 +: LSK). Deletion of Hoip treated with tamoxifen markedly impaired colony formation of LT-HSCs in vitro. Among competitive transplantation assay using bone marrow cells from Hoip fl/fl; Rosa26-CreERT mice, deletion of Hoip treated with tamoxifen following stable reconstitution 8 weeks after transplantation led to a significant reduction of Hoip-deficient chimerism compared to control in vivo (Figure A). Deletion of Hoip induced apoptosis in bone marrow cells and increased the frequency of CD34 - LSK cells in S/G2/M phase of the cell cycle. Collectively, these data indicated that Hoip is required for adult hematopoiesis.To evaluate the consequences of Hoip deletion in myeloid leukemia propagation and maintenance, we utilized aggressive murine myeloid leukemia models driven by retroviral transduction of oncogenes: MLL-AF9 and NRAS G12V-driven acute myeloid leukemia (AML) and BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) models. Deletion of Hoip in established leukemia cells treated with tamoxifen markedly impaired colony formation of leukemia cells in vitro and led to a significantly longer survival with reduced disease burden in bone marrow and spleen in vivo in both of leukemic models (Figure B,C). Deletion of Hoip also induced apoptosis in leukemia stem cells (LSCs: c-Kit + Lineage - cells) (Figure D) and increased the frequency of LSCs in S-phase of the cell cycle. Furthermore, we evaluated the contribution of HOIP to human myeloid leukemia. Knockdown of HOIP by small hairpin RNA using lentivirus in several myeloid leukemia cell lines (THP-1, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro, and increased apoptosis. LUBAC inhibitor gliotoxin impaired the growth of murine and human leukemia cells. These results indicate that Hoip is essential for propagation and maintenance in murine and human myeloid leukemia.Because LUBAC regulates canonical NF-κB signaling pathway, we evaluated protein levels of p65 phosphorylation on Ser536 (p-p65) and IκBα in leukemia cells by flow cytometric analysis. Reduction in p-p65 and inhibition of IκBα degradation were observed in Hoip-deficient leukemia cells (Figure E), indicating that Hoip plays an important role in regulation of NF-κB signaling in myeloid leukemia.Since Hoip is the central component of LUBAC and the only catalytically active subunit of E3 ubiquitin ligase, we examined the importance of each domain of Hoip for myeloid leukemia propagation using Hoip mutant isoforms. Loss of colony-forming ability of Hoip-deficient AML cells could not be rescued by retroviral transduction of the Hoip mutants with deletion of UBA domain (which is required for interaction with the other LUBAC subunits) and with deletion of RBR domain and with mutation in C879A (which lack ligase activity) (Figure F), indicating that LUBAC ligase activity and interaction with the other LUBAC subunits are critical for leukemia propagation.Thus, our data demonstrated that Hoip is essential for adult hematopoiesis and myeloid leukemia. Given that patients with myeloid leukemia have shown increased activity of NF-κB signaling, inhibition of LUBAC ligase activity could serve as a promising strategy for treating myeloid leukemia. [Display omitted] DisclosuresJimbo: Japan Society for the Promotion of Science: Research Funding. Ito: Japan Society for the Promotion of Science: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Iwama: Nissan Chemical Corporation: Research Funding. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Konuma: Japan Society for the Promotion of Science: Research Funding; The Japanese Society of Hematology: Research Funding; SGH Foundation: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

Dual roles for LUBAC signaling in thymic epithelial cell development and survival.

Thymic epithelial cells (TECs) form a unique microenvironment that orchestrates T cell differentiation and immunological tolerance. Despite the importance of TECs for adaptive immunity, there is an incomplete understanding of the signalling networks that support their differentiation and survival. We report that the linear ubiquitin chain assembly complex (LUBAC) is essential for medullary TEC (mTEC) differentiation, cortical TEC survival and prevention of premature thymic atrophy. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, severely impaired expansion of the thymic medulla and AIRE-expressing cells. Furthermore, HOIL-1-deficiency caused early thymic atrophy due to Caspase-8/MLKL-dependent apoptosis/necroptosis of cortical TECs. By contrast, deficiency in the LUBAC component, SHARPIN, caused relatively mild defects only in mTECs. These distinct roles for LUBAC components in TECs correlate with their function in linear ubiquitination, NFκB activation and cell survival. Thus, our findings reveal dual roles for LUBAC signaling in TEC differentiation and survival.

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

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.

Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling.

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF-κB reporter relative to wild-type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated HoipK778R/K778R knock-in mice, which show no overt developmental phenotypes; however, in response to TNF, HoipK778R/K778R mouse embryonic fibroblasts display mildly suppressed NF-κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF-induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in HoipK778R/K778R mice, which is rescued by knockout of TNFR1. We propose that site-specific ubiquitination of HOIP regulates a LUBAC-dependent switch between survival and apoptosis in TNF signaling.

SHARPIN stabilizes β-catenin through a linear ubiquitination-independent manner to support gastric tumorigenesis.

Aberrant activation of Wnt/β-catenin signaling by dysregulated post-translational protein modifications, especially ubiquitination is causally linked to cancer development and progression. Although Lys48-linked ubiquitination is known to regulate Wnt/β-catenin signaling, it remains largely obscure how other types of ubiquitination, such as linear ubiquitination governs its signaling activity. The expression and regulatory mechanism of linear ubiquitin chain assembly complex (LUBAC) on Wnt/β-catenin signaling was examined by immunoprecipitation, western blot and immunohistochemical staining. The ubiquitination status of β-catenin was detected by ubiquitination assay. The impacts of SHARPIN, a core component of LUBAC on malignant behaviors of gastric cancer cells were determined by various functional assays in vitro and in vivo. Unlike a canonical role in promoting linear ubiquitination, SHARPIN specifically interacts with β-catenin to maintain its protein stability. Mechanistically, SHARPIN competes with the E3 ubiquitin ligase β-Trcp1 for β-catenin binding, thereby decreasing β-catenin ubiquitination levels to abolish its proteasomal degradation. Importantly, SHARPIN is required for invasiveness and malignant growth of gastric cancer cells in vitro and in vivo, a function that is largely dependent on its binding partner β-catenin. In line with these findings, elevated expression of SHARPIN in gastric cancer tissues is associated with disease malignancy and correlates with β-catenin expression levels. Our findings reveal a novel molecular link connecting linear ubiquitination machinery and Wnt/β-catenin signaling via SHARPIN-mediated stabilization of β-catenin. Targeting the linear ubiquitination-independent function of SHARPIN could be exploited to inhibit the hyperactive β-catenin signaling in a subset of human gastric cancers.

Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP

The ubiquitylation of NF-κB essential modulator (NEMO) is part of the intracellular immune signalling pathway. Monoubiquitylated NEMO is required for exploring the mechanism of NEMO linear ubiquitylation by LUBAC (linear ubiquitin chain assembly complex), but is not accessible by biological techniques. Here we perform the chemical ubiquitylation of NEMO using a ligation auxiliary, which only requires a two-step synthesis, and is easily installed onto the lysine side-chain. Chemical ligation occurs directly on the lysine ε amine and remains efficient below pH 7. We show that ubiquitylated NEMO has similar affinity to linear di-ubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesised NEMOCoZi-Ub is accepted as a substrate for linear extension by the (RING-Between-RING) RBR domain of HOIL-1-interacting protein (HOIP) alone. Our results indicate that NEMO linear ubiquitylation consists of two-steps, an initial priming event and a separate extension step requiring different LUBAC components.

A protein quality control pathway regulated by linear ubiquitination.

Neurodegenerative diseases are characterized by the accumulation of misfolded proteins in the brain. Insights into protein quality control mechanisms to prevent neuronal dysfunction and cell death are crucial in developing causal therapies. Here, we report that various disease-associated protein aggregates are modified by the linear ubiquitin chain assembly complex (LUBAC). HOIP, the catalytic component of LUBAC, is recruited to misfolded Huntingtin in a p97/VCP-dependent manner, resulting in the assembly of linear polyubiquitin. As a consequence, the interactive surface of misfolded Huntingtin species is shielded from unwanted interactions, for example with the low complexity sequence domain-containing transcription factor Sp1, and proteasomal degradation of misfolded Huntingtin is facilitated. Notably, all three core LUBAC components are transcriptionally regulated by Sp1, linking defective LUBAC expression to Huntington's disease. In support of a protective activity of linear ubiquitination, silencing of OTULIN, a deubiquitinase with unique specificity for linear polyubiquitin, decreases proteotoxicity, whereas silencing of HOIP has the opposite effect. These findings identify linear ubiquitination as a protein quality control mechanism and hence a novel target for disease-modifying strategies in proteinopathies.

LUBAC and ABIN-1 Modulate TRAIL-Based NF-κB Induction in Human Embryonic Kidney 293 Cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known to activate the canonical NF-κB pathway similar to TNF. The exact mechanism of the entire signaling cascade is still under investigation. The involvement of linear ubiquitylation as upregulating component has already been shown recently in some cell lines, but not in human embryonic kidney 293 (HEK293) cells. The downregulating function of the ABIN-1 (A20 binding and inhibitor of NF-κB) as linear ubiquitylation antagonist has been shown in combination with some NF-κB-inducing pathways, but not with TRAIL. We performed luciferase and western blot assays using HEK293 cells stimulated with either TRAIL (or TNF as a control) to analyze the involvement of linear ubiquitin chain assembly complex (LUBAC) components and the impact of ABIN-1 and ABIN-1-MAD (truncated form without A20 binding site) on NF-κB signaling. For overexpression experiments, we added plasmids of ABIN-1 and ABIN-1-MAD or LUBAC components HOIP, HOIL-1, or SHARPIN (single and combinations). For downregulation experiments five pairs of either SHARPIN, HOIL-1, or HOIP targeting miRNAs or one miRNA for ABIN-1 were designed and added. ABIN-1 and its truncated form ABIN-1-MAD reduced the NF-κB induction significantly indicating its involvement as antagonist (independent of deubiquitinase A20) of linear ubiquitylation in TRAIL-induced NF-κB signaling. In opposition, knockdown of ABIN-1 using a specific ABIN-1 miRNA led a clear increase of NF-κB signaling. Addition of single LUBAC components or combinations (except for SHARPIN with HOIL-1) resulted in clearly stronger NF-κB inductions. MiRNAs targeting LUBAC components significantly reduced NF-κB activation. Thus, in HEK293 cells linear ubiquitylation by LUBAC critically upregulates and ABIN-1 downregulates TRAIL-induced NF-κB signaling and may be interesting targets for future pathological therapies.

LUBAC-mediated signals in thymic epithelial cells prevent early thymic atrophy and immunodeficiency

Abstract Thymic epithelial cells (TECs) govern the differentiation of haematopoietic precursors into functional T cells. Defects in this process can lead to immunodeficiency and autoimmune disease. Distinct signals from members of the TNF receptor superfamily have key roles in TEC proliferation and differentiation, yet it remains unclear how these stimuli are integrated to support thymus function. We find that components of the linear ubiquitin chain assembly complex (LUBAC) that act downstream of TNFR signaling to regulate NFkB activity are essential for TEC homeostasis and thymic function. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, induced early thymic atrophy shortly after birth, with severe loss of tissue in 2-month-old adult mice attended by gross defects in peripheral T cell populations. The requirement for HOIL-1 or HOIP extended to all major TEC subsets, including those expressing the autoimmune regulator, AIRE. In contrast, loss of a third LUBAC component in SHARPIN-deficient mice (Shcpdm/cpdm) had a milder impact, reducing TECs in the thymic medulla that require CD40 signals for their maintenance. Ablation of TNF or Caspase 8 and RIPK3 in Shcpdm/cpdm mice failed to rescue these defects, indicating that CD40/SHARPIN signals are not required to prevent TNF-induced cell death. These differential roles for LUBAC components in TEC correlate with their function in linear ubiquitylation and reveal a critical signaling hub in the maintenance of the thymic microenvironment

LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis.

The Linear Ubiquitin chain Assembly Complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR11. Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, due 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. By creating HOIL-1-deficient mice, we here show that HOIL-1 is, however, as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is LUBAC’s catalytically active component, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1-signalling complex (TNFR1-SC), 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-/- embryos, yet only combined loss of Caspase-8 with MLKL results in viable HOIL-1-deficient mice. Interestingly, Ripk3-/-Caspase-8-/-Hoil-1-/- embryos die at late-gestation due 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 unveil that, when LUBAC and Caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in foetal haematopoiesis.

The E3 ubiquitin ligases HOIP and cIAP1 are recruited to the TNFR2 signaling complex and mediate TNFR2-induced canonical NF-κB signaling

Tumor Necrosis Factor (TNF) is a proinflammatory cytokine that elicits its action by binding to two cell surface TNF receptors (TNFR), TNFR1 and TNFR2, which are expressed by many different cell types. Stimulation of TNFR1 activates canonical NF-κB signaling, leading to the NF-κB dependent expression of a large number of genes. Canonical NF-κB signaling requires the assembly of a TNFR1 signaling complex at the cell membrane, whose formation is regulated by different protein ubiquitination events. In this context, recruitment of the Linear Ubiquitin Chain Assembly Complex (LUBAC) to TNFR1 plays an important role by mediating M1-linked polyubiquitination of specific NF-κB signaling proteins. In contrast to TNFR1, much less is known about the role of ubiquitination in TNFR2 signaling. Here we demonstrate that specific TNFR2 stimulation rapidly triggers M1- and K63-linked polyubiquitination at the TNFR2 signaling complex. In agreement, TNFR2 stimulation induces the recruitment of HOIP, a LUBAC component and the only known E3 ubiquitin ligase for M1-polyubiquitination, to the TNFR2 signaling complex. Also cIAP1, a E3 ubiquitin ligase able to modify proteins with K63-polyubiquitin chains, was recruited to the TNFR2 signaling complex. Treatment of cells with a cIAP antagonist inhibited the recruitment of HOIP and prevented HOIP-mediated M1-ubiquitination of the TNFR2 signaling complex, indicating that HOIP recruitment to the TNFR2 relies on cIAPs. Finally, we show that both HOIP and cIAP1 are required for TNFR2-induced canonical NF-κB activation. Together, our findings demonstrate an important role for M1- and K63-linked polyubiquitination in TNFR2 signaling.

Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis.

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.

--LUBAC deficiency perturbs TLR3 signaling to cause immunodeficiency and autoinflammation.

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.

Mutually Exclusive Roles of SHARPIN in Integrin Inactivation and NF-κB Signaling.

SHANK-associated RH domain interactor (SHARPIN) inhibits integrins through interaction with the integrin α-subunit. In addition, SHARPIN enhances nuclear factor-kappaB (NF-κB) activity as a component of the linear ubiquitin chain assembly complex (LUBAC). However, it is currently unclear how regulation of these seemingly different roles is coordinated. Here, we show that SHARPIN binds integrin and LUBAC in a mutually exclusive manner. We map the integrin binding site on SHARPIN to the ubiquitin-like (UBL) domain, the same domain implicated in SHARPIN interaction with LUBAC component RNF31 (ring finger protein 31), and identify two SHARPIN residues (V267, L276) required for both integrin and RNF31 regulation. Accordingly, the integrin α-tail is capable of competing with RNF31 for SHARPIN binding in vitro. Importantly, the full SHARPIN RNF31-binding site contains residues (F263A/I272A) that are dispensable for SHARPIN-integrin interaction. Importantly, disrupting SHARPIN interaction with integrin or RNF31 abolishes SHARPIN-mediated regulation of integrin or NF-κB activity, respectively. Altogether these data suggest that the roles of SHARPIN in inhibiting integrin activity and supporting linear ubiquitination are (molecularly) distinct.

HOIP Deficiency Causes Embryonic Lethality by Aberrant TNFR1-Mediated Endothelial Cell Death

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.