Biochemical and functional characterisation of the Linear Ubiquitin Chain Assembly Complex (LUBAC) as a component of the TNF-Receptor 1 signalling complex

Tumour necrosis factor (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 lead to induction of proinflammatory cytokines, cell proliferation, differentiation or cell death. Most of the pleiotropic effects of TNF are mediated by the death-domain (DD) containing TNF-R1. Ligand-induced trimerisation of TNF-R1 leads to the formation of an intracellular multiprotein complex, the TNF-R1 signalling complex (TNF-RSC). To be able to comprehend the complex network of signalling pathways emanating from TNF-R1, the TNF-RSC and its composition need to be understood at the molecular level. Using a modified tandem affinity purification 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-kB activation following TNF stimulation. In this thesis, it could be demonstrated that the stimulation-dependent recruitment of LUBAC to the TNF-RSC is impaired in cIAP1/2-deficient mouse embryonic fibroblast (MEF) cell lines. Furthermore, it was shown 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. It was also found that LUBAC enhances NEMO interaction with the TNF-RSC, that it stabilises this protein complex and that LUBAC is required for efficient TNF-induced activation of NF-kB and JNK, resulting in apoptosis inhibition. Finally, it is shown in this thesis that the catalytic activity of LUBAC is required for stabilisation of the TNF-RSC, thereby adding a novel form of ubiquitin linkage to 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

Double in situ hybridization in combination with digital image analysis: A new approach to study interphase chromosome topography

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far

Reproducibility of preclinical data: one man's poison is another man's meat

Limited reproducibility of preclinical data is increasingly discussed in the literature. Failure of drug devel-opment programs due to lack of clinical efficacy is also of growing concern. The two phenomena may share an important root cause — a lack of robustness in preclinical research. Such a lack of robustness can be a relevant cause of fail-ure in translating preclinical findings into clinical efficacy and hence attrition, and exaggerated cost in drug develop-ment. Apart from the study design and data analysis factors (e.g., insufficient sample sizes, failure to implement blind-ing, and randomization), heterogeneity among experimental models (e.g., animal strains) and the conditions of the study used between different laboratories is a major contributor to the lacking of robustness of research findings. The flipside of this coin is that the understanding of the causes of heterogeneity across experimental models may lead to the identification of relevant factors for defining the responder populations. Thus, this heterogeneity within preclinical find-ings could be an asset, rather than an obstacle, for precision medicine. To enable this paradigm shift, several steps need to be taken to identify conditions under which drugs do not work. An improved granularity in the reporting of preclini-cal studies is central among them (i.e., details about the study design, experimental conditions, quality of tools and rea-gents, validation of assay conditions, etc.). These actions need to be discussed jointly by the research communities in-terested in preclinical data robustness and precision medicine. Thus, we propose that a lack of robustness due to the heterogeneity across models and conditions of the study is not necessarily a liability for biomedical research but can be transformed into an asset of precision medicine

Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling

It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the "master" protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1-dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligase-inactive TRAF6 mutants. The IL-1-induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligase-inactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKL-induced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity

Lys63/Met1-hybrid ubiquitin chains are commonly formed during the activation of innate immune signalling.

AbstractWe have reported previously that activation of the MyD88-signaling network rapidly induces the formation of hybrid ubiquitin chains containing both Lys63-linked and Met1-linked ubiquitin (Ub) oligomers, some of which are attached covalently to Interleukin Receptor Associated kinase 1. Here we show that Lys63/Met1-Ub hybrids are also formed rapidly when the TNFR1/TRADD, TLR3/TRIF- and NOD1/RIP2-signaling networks are activated, some of which are attached covalently to Receptor-Interacting Protein 1 (TNFR1 pathway) or Receptor-Interacting Protein 2 (NOD1 pathway). These observations suggest that the formation of Lys63/Met1-Ub hybrids are of general significance for the regulation of innate immune signaling systems, and their potential roles in vivo are discussed. We also report that TNFα induces the attachment of Met1-linked Ub chains directly to TNF receptor 1, which do not seem to be attached covalently to Lys63-linked or other types of ubiquitin chain

PEERS - an open science “Platform for the Exchange of Experimental Research Standards” in biomedicine

Funding The PEERS Consortium is currently funded by Cohen Veterans Bioscience Ltd and grants COH-0011 from Steven A. Cohen. Acknowledgements We would like to thank IJsbrand Jan Aalbersberg, Natasja de Bruin, Philippe Chamiot-Clerc, Anja Gilis, Lieve Heylen, Martine Hofmann, Patricia Kabitzke, Isabel Lefevre, Janko Samardzic, Susanne Schiffmann and Guido Steiner for their valuable input and discussions during the conceptualization of PEERS and the initial phase of the project.Peer reviewedPublisher PD

Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages

Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4-activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4-activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization

The anti-inflammatory drug BAY 11-7082 suppresses the MyD88-dependent signalling network by targeting the ubiquitin system

The compound BAY 11-7082 inhibits IκBα [inhibitor of NF-κB (nuclear factor κB)α] phosphorylation in cells and has been used to implicate the canonical IKKs (IκB kinases) and NF-κB in >350 publications. In the present study we report that BAY 11-7082 does not inhibit the IKKs, but suppresses their activation in LPS (lipopolysaccharide)-stimulated RAW macrophages and IL (interleukin)-1-stimulated IL-1R (IL-1 receptor) HEK (human embryonic kidney)-293 cells. BAY 11-7082 exerts these effects by inactivating the E2-conjugating enzymes Ubc (ubiquitin conjugating) 13 and UbcH7 and the E3 ligase LUBAC (linear ubiquitin assembly complex), thereby preventing the formation of Lys(63)-linked and linear polyubiquitin chains. BAY 11-7082 prevents ubiquitin conjugation to Ubc13 and UbcH7 by forming a covalent adduct with their reactive cysteine residues via Michael addition at the C(3) atom of BAY 11-7082, followed by the release of 4-methylbenzene-sulfinic acid. BAY 11-7082 stimulated Lys(48)-linked polyubiquitin chain formation in cells and protected HIF1α (hypoxia-inducible factor 1α) from proteasomal degradation, suggesting that it inhibits the proteasome. The results of the present study indicate that the anti-inflammatory effects of BAY 11-7082, its ability to induce B-cell lymphoma and leukaemic T-cell death and to prevent the recruitment of proteins to sites of DNA damage are exerted via inhibition of components of the ubiquitin system and not by inhibiting NF-κB

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

Clinical Use and Therapeutic Potential of IVIG/SCIG, Plasma-Derived IgA or IgM, and Other Alternative Immunoglobulin Preparations

Intravenous and subcutaneous immunoglobulin preparations, consisting of IgG class antibodies, are increasingly used to treat a broad range of pathological conditions, including humoral immune deficiencies, as well as acute and chronic inflammatory or autoimmune disorders. A plethora of Fab- or Fc-mediated immune regulatory mechanisms has been described that might act separately or in concert, depending on pathogenesis or stage of clinical condition. Attempts have been undertaken to improve the efficacy of polyclonal IgG preparations, including the identification of relevant subfractions, mild chemical modification of molecules, or modification of carbohydrate side chains. Furthermore, plasma-derived IgA or IgM preparations may exhibit characteristics that might be exploited therapeutically. The need for improved treatment strategies without increase in plasma demand is a goal and might be achieved by more optimal use of plasma-derived proteins, including the IgA and the IgM fractions. This article provides an overview on the current knowledge and future strategies to improve the efficacy of regular IgG preparations and discusses the potential of human plasma-derived IgA, IgM, and preparations composed of mixtures of IgG, IgA, and IgM