The role of HOIL-1, HOIP and SHARPIN in immune signalling

Members of the tumour necrosis factor receptor (TNFR) superfamily, the interleukin-1 Receptor (IL-1R), the Toll-like Receptor (TLR) and the NOD-like receptor (NLRs) families play crucial roles in the initiation of innate immune responses. Even though the stimulation of these different receptors is triggered by upstream signalling components which are largely receptor-specific, their signalling cascades generally converge in the activation of both mitogen-activated protein kinases (MAPKs) and the inhibitor of nuclear factor–B (IB) kinases (IKKs). The activation of MAPKs and IKKs are crucial events in the signalling cascades of TNFR-, IL-1R-, TLR- and NLR family members and result in the activation of distinct transcription factors such as AP-1 and NF-B, respectively. In order to gain a more comprehensive picture of the signalling components associated with TNFR1 our group developed a modified tandem affinity purification (moTAP) strategy. This led to the identification of three novel components of the native TNFR1 signalling complex (TNFRSC): HOIL-1, HOIP and SHARPIN. Together, they form the “linear ubiquitin chain assembly complex” (LUBAC), a tripartite E3 ligase complex which generates linear ubiquitin chains. LUBAC activity is required for efficient TNF-mediated activation of NF-B and JNK by linearly ubiquitinating NEMO and RIP1 in the TNF-RSC. Mutation of the SHARPIN-encoding gene in mice results in chronic proliferative dermatitis (cpdm). The cpdm phenotype is characterised by inflammatory skin lesions and defective lymphoid organogenesis. In this thesis it is shown that mouse embryonic fibroblasts (MEFs) and primary keratinocytes generated from cpdm mice showed impaired activation of the MAPK- and NF-B signalling pathways following stimulation by various TNF and IL-1 family members as well as by different TLR ligands. These alterations were also apparent in bone marrow-derived macrophages isolated from cpdm mice. Thereby, lack of SHARPIN exerted an inhibitory effect on gene-activatory signal transduction by these immune stimuli. Similar defects in NF- B and MAPK signalling were also observed with a ligand for the NLR family member NOD2. Cells generated from mice conditionally deficient for the other LUBAC components exhibited similar alterations in diverse innate immune signalling pathways. These results imply that SHARPIN and HOIL-1 cannot substitute each other but instead cooperate with HOIP to generate linear ubiquitin chains in the signalling pathways that are activated by diverse immune receptors. In summary, the results of this thesis identify LUBAC and the linear ubiquitin chains it generates as previously unrecognised components of various signalling pathways which are central to the induction of immunity and regulation of inflammation

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.This work was funded by a Wellcome Trust Senior Investigator award (096831/Z/11/Z; and grant 090315 to H. Ren) and an European Research Council advanced grant (294880; H. Walczak). J. Zinngrebe received support from the Boehringer Ingelheim Fonds and N. Peltzer received funds from the Swiss National Science Foundation. B.J. Ferguson is supported by an Isaac Newton Trust/Wellcome Trust ISSF/University of Cambridge research grant. B. Dome received support from the Hungarian Scientific Research Fund (OTKA-K108465)

Recruitment of the Linear Ubiquitin Chain Assembly Complex Stabilizes the TNF-R1 Signaling Complex and Is Required for TNF-Mediated Gene Induction

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-κB 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. © 2009 Elsevier Inc. All rights reserved

Inhibition of ADAM17 impairs endothelial cell necroptosis and blocks metastasis

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 therapie

­­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

Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8

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

The linear ubiquitin chain assembly complex (LUBAC) forms part of the TNF-R1 signalling complex and is required for effective TNF-induced gene induction and prevents TNF-induced apoptosis

For antigen and various cytokine receptor-mediated signalling events, ubiquitination is a fundamental regulatory mechanism implicated in the different signalling pathways emanating from these receptors. Since the discovery of the importance of lysine 48-linked ubiquitin chains for NF-?B activation and the finding that the ubiquitin ligases TRAF2 and cIAP1/2 are recruited to the TNF-RSC, it became clear that the activation of TNF-induced signalling pathways depends strongly on the ubiquitin system. TNF plays a critical role in inflammatory processes and is involved in the regulation of immune responses. Depending on the cellular context, TNF initiates a complex cascade of signalling events that can result in induction of proinflammatory cytokines, cell proliferation, differentiation or cell death. Ligand-induced trimerisation of TNF-R1 leads to the formation of a multi-protein complex, the TNFR1 signalling complex (TNF-RSC). To be able to understand the multifaceted regulatory functions of the ubiquitin network and to comprehend the complex interplay of signalling pathways emanating from TNF-R1, the TNF-RSC and its composition need to be understood at the molecular level. Therefore we newly developed a modified tandem affinity purification (moTAP) procedure which allowed us to physically determine the molecular composition of the TNF-RSC in an unbiased manner. Using the moTAP approach, HOIL-1 and HOIP were identified as two novel, functional components of the native TNF-RSC. Together they were shown to form a linear ubiquitin chain assembly complex (LUBAC), catalysing the formation of linear head-to-tail ubiquitin chains. LUBAC mediates ubiquitination of NEMO with linear ubiquitin chains, required for efficient NF-?B activation following TNF stimulation. We show that the stimulation-dependent recruitment of LUBAC to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2-, but not in RIP1- and NEMO-deficient mouse embryonic fibroblast (MEF) cell lines. Furthermore, we demonstrate that the E3 ligase activity of cIAPs, but not of TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. This result, together with the ability of HOIL-1 and HOIP to bind polyubiquitin chains of various linkage types, suggests that LUBAC is recruited to the TNF-RSC via cIAP-generated ubiquitin chains. LUBAC enhances NEMO interaction with the TNF-RSC, stabilises this protein complex, and is required for efficient TNF-induced activation of NF-?B and JNK, resulting in apoptosis inhibition. Finally, we show that the activity of LUBAC is required for stabilisation of the TNF-RSC, thereby adding a third form of ubiquitin linkage to the control of TNF signalling. The identification of HOIL-1 and HOIP as functional constituents of the TNF-RSC provides evidence that LUBAC is an important regulator at the apex of TNF-induced signalling cascades and increases the combinatorial complexity of ubiquitin modifications within this receptor complex. \uc2\ua9 2011 Springer Science+Business Media, LLC

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