Fabrice Agou

Complementation Cloning of NEMO, a Component of the IκB Kinase Complex Essential for NF-κB Activation

Abstract We have characterized a flat cellular variant of HTLV-1 Tax-transformed rat fibroblasts, 5R, which is unresponsive to all tested NF-κB activating stimuli, and we report here its genetic complementation. The recovered full-length cDNA encodes a 48 kDa protein, NEMO ( N F-κB E ssential MO dulator), which contains a putative leucine zipper motif. This protein is absent from 5R cells, is part of the high molecular weight IκB kinase complex, and is required for its formation. In vitro, NEMO can homodimerize and directly interacts with IKK-2. The NEMO cDNA was also able to complement another NF-κB–unresponsive cell line, 1.3E2, in which the protein is also absent, allowing us to demonstrate that this factor is required not only for Tax but also for LPS, PMA, and IL-1 stimulation of NF-κB activity.

The p43 Component of the Mammalian Multi-synthetase Complex Is Likely To Be the Precursor of the Endothelial Monocyte-activating Polypeptide II Cytokine

p43 is one of the three auxiliary components invariably associated with nine aminoacyl-tRNA synthetases as a multienzyme complex ubiquitous to all eukaryotic cells from flies to humans. The cDNA encoding the hamster protein was isolated by using mixed oligonucleotides deduced from peptide sequences. The 359-amino acid protein is the hamster homologue of the recently reported murine and human EMAP II cytokine implicated in a variety of inflammatory disorders. The sequence of several proEMAP II proteins suggests that the p43 component of the complex is the precursor of the active mature cytokine after cleavage at a conserved Asp residue. The COOH-terminal moiety of p43 is also homologous to polypeptide domains found in bacterial methionyl- or phenylalanyl-tRNA synthetases and in the yeast Arc1p/G4p1 protein that associates with yeast methionyl-tRNA synthetase. Our results implicate the COOH-terminal moiety of p43 as a RNA binding domain. In the native state, as a component of the multisynthetase complex, p43 may be required for tRNA channeling and, after proteolytic processing occurring in tumor cells, would acquire inflammatory properties possibly related to apoptosis. The release of a truncated p43 from the complex could be involved in mediation of the signaling of tumor cells and stimulation of an acute inflammatory response.

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.

TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO–IKK supramolecular structures

The mechanism of NEMO recruitment into supramolecular complexes and its dependence on ubiquitination differs in response to the proinflammatory cytokines TNF and IL-1.

The Zinc Finger of NEMO Is a Functional Ubiquitin-binding Domain

NEMO (NF-κB essential modulator) is a regulatory protein essential to the canonical NF-κB signaling pathway, notably involved in immune and inflammatory responses, apoptosis, and oncogenesis. Here, we report that the zinc finger (ZF) motif, located in the regulatory C-terminal half of NEMO, forms a specific complex with ubiquitin. We have investigated the NEMO ZF-ubiquitin interaction and proposed a structural model of the complex based on NMR, fluorescence, and mutagenesis data and on the sequence homology with the polymerase η ubiquitin-binding zinc finger involved in DNA repair. Functional complementation assays and in vivo pull-down experiments further show that ZF residues involved in ubiquitin binding are functionally important and required for NF-κB signaling in response to tumor necrosis factor-α. Thus, our findings indicate that NEMOZFisa bona fide ubiquitin-binding domain of the ubiquitin-binding zinc finger type.

IκB kinase overcomes PI3K/Akt and ERK/MAPK to control FOXO3a activity in acute myeloid leukemia

The FOXO transcription factors are involved in multiple signaling pathways and have tumor-suppressor functions. In acute myeloid leukemia (AML), deregulation of oncogenic kinases, including Akt, extra-signal-regulated kinase, or IκB kinase, is frequently observed, which may potentially inactivate FOXO activity. We therefore investigated the mechanism underlying the regulation of FOXO3a, the only FOXO protein constantly expressed in AML blast cells. We show that in both primary AML samples and in a MV4-11/FOXO3a-GFP cell line, FOXO3a is in a constant inactive state due to its cytoplasmic localization, and that neither PI3K/Akt nor extra-signal-regulated kinase-specific inhibition resulted in its nuclear translocation. In contrast, the anti-Nemo peptide that specifically inhibits IKK activity was found to induce FOXO3a nuclear localization in leukemic cells. Furthermore, an IKK-insensitive FOXO3a protein mutated at S⁶⁴⁴ translocated into the nucleus and activated the transcription of the Fas-L and p21(Cip1) genes. This, in turn, inhibited leukemic cell proliferation and induced apoptosis. These results thus indicate that IKK activity maintains FOXO3a in the cytoplasm and establishes an important role of FOXO3a inactivation in the proliferation and survival of AML cells. The restoration of FOXO3a activity by interacting with its subcellular distribution may thus represent a new attractive therapeutic strategy for AML.

Solution structure of NEMO zinc finger and impact of an anhidrotic ectodermal dysplasia with immunodeficiency-related point mutation.

The regulatory NEMO (NF-kappaB essential modulator) protein has a crucial role in the canonical NF-kappaB signaling pathway notably involved in immune and inflammatory responses, apoptosis and oncogenesis. The regulatory domain is located in the C-terminal half of NEMO and contains a classical CCHC-type zinc finger (ZF). We have investigated the structural and functional effects of a cysteine to phenylalanine point mutation (C417F) in the ZF motif, identified in patients with anhidrotic ectodermal dysplasia with immunodeficiency. The solution structures of the wild type and mutant ZF were determined by NMR. Remarkably, the mutant adopts a global betabetaalpha fold similar to that of the wild type and retains thermodynamic stability, i.e., the ability to bind zinc with a native-like affinity, although the last zinc-chelating residue is missing. However, the mutation induces enhanced dynamics in the motif and leads to an important loss of stability. A detailed analysis of the wild type solution structure and experimental evidences led to the identification of two possible protein-binding surfaces that are largely destabilized in the mutant. This is sufficient to alter NEMO function, since functional complementation assays using NEMO-deficient pre-B and T lymphocytes show that full-length C417F pathogenic NEMO leads to a partial to strong defect in LPS, IL-1beta and TNF-alpha-induced NF-kappaB activation, respectively, as compared to wild type NEMO. Altogether, these results shed light onto the role of NEMO ZF as a protein-binding motif and show that a precise structural integrity of the ZF should be preserved to lead to a functional protein-recognition motif triggering full NF-kappaB activation.

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.

A point mutation in NEMO associated with anhidrotic ectodermal dysplasia with immunodeficiency pathology results in destabilization of the oligomer and reduces lipopolysaccharide- and tumor necrosis factor-mediated NF-kappa B activation.

The NEMO (NF-κB essential modulator) protein plays a crucial role in the canonical NF-κB pathway as the regulatory component of the IKK (IκB kinase) complex. The human disease anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has been recently linked to mutations in NEMO. We investigated the effect of an alanine to glycine substitution found in the NEMO polypeptide of an EDA-ID patient. This pathogenic mutation is located within the minimal oligomerization domain of the protein, which is required for the IKK activation in response to diverse stimuli. The mutation does not dramatically change the native-like state of the trimer, but temperature-induced unfolding studied by circular dichroism showed that it leads to an important loss in the oligomer stability. Furthermore, fluorescence studies showed that the tyrosine located in the adjacent zinc finger domain, which is possibly required for NEMO ubiquitination, exhibits an alteration in its spectral properties. This is probably due to a conformational change of this domain, providing evidence for a close interaction between the oligomerization domain and the zinc finger. In addition, functional complementation assays using NEMO-deficient pre-B and T lymphocytes showed that the pathogenic mutation reduced TNF-α and LPS-induced NF-κB activation by altering the assembly of the IKK complex. Altogether, our findings provide understanding as to how a single point mutation in NEMO leads to the observed EDA-ID phenotype in relation to the NEMO-dependent mechanism of IKK activation.

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.