Elisabeth Fontan

NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain

An important property of NEMO, the core element of the IKK complex involved in NF‐κB activation, resides in its ability to specifically recognize poly‐ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the C‐terminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63‐linked chains, we demonstrate that together they form a bipartite high‐affinity K63‐specific ubiquitin‐binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C‐terminal half of NEMO is to specifically bind K63‐linked poly‐ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly‐ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.

NEMO oligomerization in the dynamic assembly of the IκB kinase core complex

NF‐κB essential modulator (NEMO) plays an essential role in the nuclear factor κB (NF‐κB) pathway as a modulator of the two other subunits of the IκB kinase (IKK) complex, i.e. the protein kinases, IKKα and IKKβ. Previous reports all envision the IKK complex to be a static entity. Using glycerol‐gradient ultracentrifugation, we observed stimulus‐dependent dynamic IKK complex assembly. In wild‐type fibroblasts, the kinases and a portion of cellular NEMO associate in a 350‐kDa high‐molecular‐mass complex. In response to constitutive NF‐κB stimulation by Tax, we observed NEMO recruitment and oligomerization to a shifted high‐molecular‐mass complex of 440 kDa which displayed increased IKK activity. This stimulus‐dependent oligomerization of NEMO was also observed using fluorescence resonance energy transfer after a transient pulse with interleukin‐1β. In addition, fully activated, dimeric kinases not bound to NEMO were detected in these Tax‐activated fibroblasts. By glycerol gradient ultracentrifugation, we also showed that: (a) in fibroblasts deficient in IKKα and IKKβ, NEMO predominantly exists as a monomer; (b) in NEMO‐deficient fibroblasts, IKKβ dimers are present that are less stable than IKKα dimers. Intriguingly, in resting Rat‐1 fibroblasts, 160‐kDa IKKα–NEMO and IKKβ–NEMO heterocomplexes were observed as well as a significant proportion of NEMO monomer. These results suggest that most NEMO molecules do not form a tripartite IKK complex with an IKKα–IKKβ heterodimer as previously reported in the literature but, instead, NEMO is able to form a complex with the monomeric forms of IKKα and IKKβ.

DARPin-assisted crystallography of the CC2-LZ domain of NEMO reveals a coupling between dimerization and ubiquitin binding.

NEMO is an integral part of the IkappaB kinase complex and serves as a molecular switch by which the NF-kappaB signaling pathway can be regulated. Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the regulatory CC2-LZ domain, were shown to be key features governing NEMO function, but the relationship between these two activities remains unclear. In this study, we solved the structure of this domain in complex with a designed ankyrin repeat protein, which helps its crystallization. We generated several NEMO mutants in this domain, including those associated with human diseases incontinentia pigmenti and immunodeficiency with or without anhidrotic ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation experiments were used to evaluate the dimerization properties of these mutants. A fluorescence-based assay was developed, as well, to quantify the interaction to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was investigated for the full-length protein. We show that a proper folding of the ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer is required but not sufficient for efficient interaction with poly-Ub. In addition, we show that binding to poly-Ub and, to a lesser extent, to monoubiquitin increases the stability of the NOA coiled-coil dimer. Collectively, these data provide structural insights into how several pathological mutations within and outside of the CC2-LZs NOA ubiquitin binding site affect IkappaB kinase activation in the NF-kappaB signaling pathway.

Enteral immunization with attenuated recombinant Listeria monocytogenes as a live vaccine vector: organ-dependent dynamics of CD4 T lymphocytes reactive to a Leishmania major tracer epitope.

ABSTRACT Listeria monocytogenes is considered as a potential live bacterial vector, particularly for the induction of CD8 T cells. The CD4 T-cell immune response triggered after enteral immunization of mice has not yet been thoroughly characterized. The dynamics of gamma interferon (IFN-γ)- and interleukin-4 (IL-4)-secreting CD4 T cells were analyzed after priming through intragastric delivery of an attenuated ΔactA recombinant L. monocytogenes strain expressing the Leishmania major LACK protein; a peptide of this protein, LACK158-173 peptide (pLACK), is a well-characterized CD4 T-cell target in BALB/c mice. Five compartments were monitored: Peyers patches, mesenteric lymph nodes (MLN), spleen, liver, and blood. A single intragastric inoculation of ΔactA-LACK-LM in BALB/c mice led to colonization of the MLN and spleen at a significant level for at least 3 days. Efficient priming of IFN-γ-secreting pLACK-reactive CD4 T cells was observed in all tested compartments. Interestingly, IL-4-secreting pLACK-reactive CD4 T cells were detectable at day 6 or 7 only in blood and liver. The absence of translocation of viable bacteria through the intestinal epithelium after further ΔactA-LACK-LM inoculations was concomitant with the absence of an increase in the level of IFN-γ secreted by the MLN, blood, and splenic pLACK-reactive Th1 T cells, although the levels remained significantly above the basal level. No change in this population size was detected in the spleen. However, an increase in the number of intragastric inoculations had a clinical beneficial effect in L. major-infected BALB/c mice. L. monocytogenes thus presents the potential of an efficient vector for induction of CD4 T cells when administered by the enteral route.

Direct inhibition of NF-κB activation by peptide targeting the NOA ubiquitin binding domain of NEMO

Aberrant and constitutive NF-κB activation are frequently reported in numerous tumor types, making its inhibition an attractive target for the treatment of certain cancers. NEMO (NF-κB essential modulator) is the crucial component of the canonical NF-κB pathway that mediates IκB kinase (IKK) complex activation. IKK activation resides in the ability of the C-terminal domain of NEMO to properly dimerize and interact with linear and K63-linked polyubiquitin chains. Here, we have identified a new NEMO peptide inhibitor, termed UBI (ubiquitin binding inhibitor) that derives from the NOA/NUB/UBAN ubiquitin binding site located in the CC2-LZ domain of NEMO. UBI specifically inhibits the NF-κB pathway at the IKK level in different cell types stimulated by a variety of NF-κB signals. Circular dichroïsm and fluorescence studies showed that UBI exhibits an increased α-helix character and direct, good-affinity binding to the NOA-LZ region of NEMO. We also showed that UBI targets NEMO in cells but its mode of inhibition is completely different from the previously reported LZ peptide (herein denoted NOA-LZ). UBI does not promote dissociation of NEMO subunits in cells but impairs the interaction between the NOA UBD of NEMO and polyubiquitin chains. Importantly, we showed that UBI efficiently competes with the in vitro binding of K63-linked chains, but not with linear chains. The identification of this new NEMO inhibitor emphasizes the important contribution of K63-linked chains for IKK activation in NF-κB signaling and would provide a new tool for studying the complex role of NF-κB in inflammation and cancer.

Characterisation of a Listeria monocytogenes mutant deficient in D-arabitol fermentation

We selected and analysed a Tn917-lac Listeria monocytogenes mutant deficient in D-arabitol fermentation. Comparison of the 310-aa-long translated partial sequence of the disrupted gene with known proteins showed similarity with the phosphotransferase system galactitol-specific enzyme IIC component of the alkaliphilic Bacillus halodurans (50% identity) and of Escherichia coli (36% identity). Fermentation of 18 other carbohydrates was unimpaired, suggesting the specificity of this transmembrane permease IIC for the pentitol D-arabitol. The deficiency in D-arabitol fermentation did not alter L. monocytogenes virulence in the BALB/c mouse model after intravenous and intragastric inoculations. This fully virulent mutant is a valuable tool to study L. monocytogenes oral infection, since the antibiotic resistance marker present on the Tn917-lac transposon will efficiently select L. monocytogenes against the intestinal microflora.

Two-sided ubiquitin binding of NF-κB essential modulator (NEMO) zinc finger unveiled by a mutation associated with anhidrotic ectodermal dysplasia with immunodeficiency syndrome

Background: Mutations in the NEMO ZF cause various forms of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID). Results: The NEMO ZF contains a larger ubiquitin binding surface, which is required for IKK/NF-κB activation. Conclusion: This study provides an understanding of how defective mutations of NEMO ZF in NF-κB signaling leads to the EDA-ID pathology. Significance: This study provides novel insights in the NEMO ZF-dependent mechanisms for IKK activation in NF-κB signaling. Hypomorphic mutations in the X-linked human NEMO gene result in various forms of anhidrotic ectodermal dysplasia with immunodeficiency. NEMO function is mediated by two distal ubiquitin binding domains located in the regulatory C-terminal domain of the protein: the coiled-coil 2-leucine zipper (CC2-LZ) domain and the zinc finger (ZF) domain. Here, we investigated the effect of the D406V mutation found in the NEMO ZF of an ectodermal dysplasia with immunodeficiency patients. This point mutation does not impair the folding of NEMO ZF or mono-ubiquitin binding but is sufficient to alter NEMO function, as NEMO-deficient fibroblasts and Jurkat T lymphocytes reconstituted with full-length D406V NEMO lead to partial and strong defects in NF-κB activation, respectively. To further characterize the ubiquitin binding properties of NEMO ZF, we employed di-ubiquitin (di-Ub) chains composed of several different linkages (Lys-48, Lys-63, and linear (Met-1-linked)). We showed that the pathogenic mutation preferentially impairs the interaction with Lys-63 and Met-1-linked di-Ub, which correlates with its ubiquitin binding defect in vivo. Furthermore, sedimentation velocity and gel filtration showed that NEMO ZF, like other NEMO related-ZFs, binds mono-Ub and di-Ub with distinct stoichiometries, indicating the presence of a new Ub site within the NEMO ZF. Extensive mutagenesis was then performed on NEMO ZF and characterization of mutants allowed the proposal of a structural model of NEMO ZF in interaction with a Lys-63 di-Ub chain.

Remote effects of inflammation on non-specific immunity

Following inflammation induced in mice with non-biodegradable, non-diffusible, and non-antigenic substances, host resistance is increased against bacteria, parasites and malignant cells injected at a distance from the inflammatory focus. This resistance is also increased in germ-free and nude mice. The increased resistance is correlated with (1) an increased leukopoiesis induced, at least in part, by a protein (MW = 40 kDa, pI = 5.2) which, in vitro, is able to induce the differentiation of bone-marrow cells into polymorphs; (2) the occurrence of giant cells in the granuloma which, after incubation in vitro, release an immunostimulating protein able to activate mice macrophages in vivo; (3) activation in vivo of liver and spleen macrophages following the occurrence, both in granuloma and in serum, of a protein (MW = 56 kDa, pI = 5) which, contrary to endotoxin, is heat-labile and can fully protect mice against Listeria monocytogenes. Furthermore, this protein, which is different from TNF and GM CSF, is able to activate macrophages against Lewis tumor cells. The same protein can be isolated from rat and is antigenically related to a human protein having the same biological activity.

Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti

The NF-κB pathway has critical roles in cancer, immunity and inflammatory responses. Understanding the mechanism(s) by which mutations in genes involved in the pathway cause disease has provided valuable insight into its regulation, yet many aspects remain unexplained. Several lines of evidence have led to the hypothesis that the regulatory/sensor protein NEMO acts as a biological binary switch. This hypothesis depends on the formation of a higher-order structure, which has yet to be identified using traditional molecular techniques. Here we use super-resolution microscopy to reveal the existence of higher-order NEMO lattice structures dependent on the presence of polyubiquitin chains before NF-κB activation. Such structures may permit proximity-based trans-autophosphorylation, leading to cooperative activation of the signalling cascade. We further show that NF-κB activation results in modification of these structures. Finally, we demonstrate that these structures are abrogated in cells derived from incontinentia pigmenti patients.

The anti‐tumoral activity of human glycoprotein HGP.92: A study with the mouse Lewis‐lung‐tumor cell

The ability of a purified human glycoprotein (HGP.92) to exert anti‐tumor activity was investigated in a mouse model using long‐term readout assays. In vitro, in the presence of inflammatory mouse macrophages incubated with HGP.92, the growth of the mouse Lewis‐lung‐tumor cells (3LL) was decreased. This effect was concentration‐dependent and required direct contact between tumor targets and HGP.92‐treated macrophages. In addition, if the macrophage monolayer was depleted of HGP.92 before addition of the target cells, no more cytostatic effect was observed. This anti‐tumor activity of HGP.92‐treated mouse macrophage was partially abrogated by addition of catalase in the culture medium, but not by superoxide dismutase or scavengers of the hydroxyl radical and singlet oxygen. Moreover, this tumor‐cell growth reduction was not dependent on nitric oxide. In vivo, multiple i.v. injections of HGP.92 (5 times, 3 days apart) during the first week and a half exerted significant anti‐tumor activity, as assessed by the reduction of both the number and the size of the lung nodules 3 weeks after i.v. inoculation of 3LL cells. Int. J. Cancer, 70:0–0, 1997.