Fabrice Agou - Academia.edu (original) (raw)

Papers by Fabrice Agou

FEBS Journal, Apr 10, 2007

NF-kappaB essential modulator (NEMO) plays an essential role in the nuclear factor kappaB (NF-kap... more NF-kappaB essential modulator (NEMO) plays an essential role in the nuclear factor kappaB (NF-kappaB) pathway as a modulator of the two other subunits of the IkappaB kinase (IKK) complex, i.e. the protein kinases, IKKalpha and IKKbeta. 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-kappaB 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-1beta. 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 IKKalpha and IKKbeta, NEMO predominantly exists as a monomer; (b) in NEMO-deficient fibroblasts, IKKbeta dimers are present that are less stable than IKKalpha dimers. Intriguingly, in resting Rat-1 fibroblasts, 160-kDa IKKalpha-NEMO and IKKbeta-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 IKKalpha-IKKbeta heterodimer as previously reported in the literature but, instead, NEMO is able to form a complex with the monomeric forms of IKKalpha and IKKbeta.

medRxiv (Cold Spring Harbor Laboratory), Aug 2, 2023

doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by pee... more doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

FEBS Letters, Nov 24, 1997

Journal of Biological Chemistry, Mar 1, 2006

The NEMO (NF-B essential modulator) protein plays a crucial role in the canonical NF-B pathway as... more The NEMO (NF-B essential modulator) protein plays a crucial role in the canonical NF-B pathway as the regulatory component of the IKK (IB 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-␣ and LPS-induced NF-B 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. The NF-B family of eucaryotic transcription factors plays a crucial role in immune and inflammatory responses, development, and control of apoptosis (1). In addition, several genetic diseases have been recently linked to defects in the NF-B pathway activation, and some of them are associated with mutations in the gene encoding the NF-B essential modulator (NEMO/IKK␥) protein (see Ref. 2 for a review). Amorphic mutations in NEMO cause incontinentia pigmenti, an X-linked dominant disorder with a variety of developmental abnormalities of the skin, teeth, hair, eyes, and central nervous system in heterozygous females and death in male fetuses (3). Most patients with incontinentia pigmenti carry a common DNA rearrangement resulting in truncation of the NEMO protein and complete suppression of NF-B activation (4). Other pathologies are caused by NEMO mutations that impair but do not abolish NF-B signaling from receptors required for ectodermal development and/or immune function. Among those, X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) 2 has been associated with mutations in the coding region of the NEMO gene (5-7). The EDA developmental phenotype is characterized by rare conical teeth, sparse scalp hair, frontal bossing, and the absence or rarity of sweat glands. Male subjects with EDA-ID present EDA combined with life-threatening infections of multiple sites, including the digestive and respiratory tracts and the skin, the most common infectious agents being Gram-positive and-negative pyogenic bacteria as well as mycobacteria. A NEMO stop codon mutation causes a clinically more severe syndrome associating osteopetrosis and/or lymphoedema with EDA-ID (5). Recently, it has also been shown that hypomorphic NEMO mutations could cause immunodeficiency without ectodermal dysplasia (8, 9). NEMO is part of the IKK (IB kinase) complex, which also contains the protein kinases IKK␣ and IKK␤ (10). Although NEMO does not possess a catalytic function, it is indispensable for signaling through the canonical NF-B pathway, since cells not expressing the protein are totally unresponsive to all tested stimuli (11, 12). In the canonical pathway, proinflammatory stimuli lead to the activation of the IKK complex (13). The kinases then phosphorylate the inhibitory IB proteins, which keep NF-B transcription factors inactive in the cytoplasm of resting cells. IBs are in turn polyubiquitinated and degraded by the proteasome, allowing NF-B to enter the nucleus and activate gene transcription. The activation mechanism of the IKK complex is still unclear. NEMO functions as a signaling adaptor between the upstream regulators and the kinases, and its oligomerization has been shown to be required for the activation of the complex (14-17). Several studies have shown that phosphorylation (18, 19) and ubiquitination (20-23) of NEMO may also contribute to the regulation of IKK activation, but the precise role of these post-translational modifications remains to be elucidated. * This work was supported by Association pour la Recherche sur le Cancer Grant 5795 and grants from the Cancé ropô le Ile-de-France, the Ligue Nationale Contre le Cancer (é quipe labelisé e; to A. I.), and the Fondation pour la Recherche Mé dicale (to G. C.

Journal of Molecular Biology, Apr 1, 2008

Biochemistry, Jul 17, 1998

Scientific Reports, Nov 4, 2019

Antiviral Therapy, May 1, 2011

Les aminoacyl-arnt synthetases catalysent l'aminoacylation specifique des arn de transfert. C... more Les aminoacyl-arnt synthetases catalysent l'aminoacylation specifique des arn de transfert. Cette these est une contribution a la caracterisation structurale et fonctionnelle des aminoacyl-arnt synthetases chez les eucaryotes. Celles-ci se differencient de leurs homologues bacteriennes par leur aptitude a se lier a des supports polyanioniques, a s'associer sous forme de complexes multienzymatiques ou a former des proteines multifonctionnelles. L'evolution structurale de cette famille d'enzyme est a mettre en parallele avec le haut niveau d'integration cellulaire de l'appareil de biosynthese des proteines chez les eucaryotes superieurs. L'aspartyl-arnt synthetase (asprs) a ete etudiee en tant que systeme modele pour la definition de motifs structuraux impliques dans des interactions avec d'autres composantes cellulaires. L'etude structurale, par dichroisme circulaire et rmn, d'un peptide representant l'extension polycationique amino-terminale de l'asprs de levure a montre que la neutralisation des chaines laterales des residus lysine par deprotonation a ph basique ou par formation d'un complexe de haute affinite avec un polyphosphate p#1#8 permet de structurer le tricosapeptide en helice , revelant une distribution anisotrope des residus basiques. Une telle structuration du domaine n-terminal de l'enzyme permettrait de former une surface d'interaction ideale envers des supports polyanioniques. Un role de ces extensions dans l'adressage des synthetases de levure au voisinage des sites de proteosynthese, a ainsi ete suggere. Chez les mammiferes, l'asprs fait partie d'un complexe multienzymatique de haut poids moleculaire. La grande similitude des structures primaires des asprss de levure et de mammifere a permis de modeliser les elements structuraux de l'enzyme de rat d'apres les coordonnees cristallographiques de l'enzyme de levure. La structure exon/intron du gene drs1 a ete correlee a la structure de la proteine. Cette analyse a conduit a mettre en evidence plusieurs caracteristiques essentielles de l'agencement exon/intron des genes morceles, en relation avec l'evolution des aminoacyl-arnt synthetases de classe ii. L'asprs de rat se distingue de son homologue de levure par une extension amino-terminale plus apolaire, impliquee dans son adressage au complexe multienzymatique. Afin d'etudier les proprietes d'association de cet enzyme, l'asprs recombinante native, exprimee dans le levure a partir de son adnc, a ete purifiee a homogeneite. De meme, des mutants de deletion ainsi que des variants portant des mutations ponctuelles dans le domaine n-terminal de l'enzyme, ont ete isoles. Les activites catalytiques de ces formes enzymatiques, determinees pour la reaction d'aminoacylation de l'arnt, sont indistinguables, soulignant l'independance du domaine catalytique et du domaine d'association de cet enzyme. Les proprietes cinetiques, hydrodynamiques et de fluorescence intrinseque de ces proteines recombinantes ont permis d'etablir une correlation entre activite enzymatique et etat dimerique de la proteine. Nous montrons que le domaine n-terminal de l'asprs de rat contribue a la stabilisation du dimere. Les differentes formes enzymatiques ont ete testees pour leur capacite a s'associer au complexe multienzymatique. Un test d'echange enzyme libre/enzyme associe a ete developpe, permettant de mesurer l'affinite relative de ces mutants vis-a-vis du complexe. Les resultats montrent que le segment comprenant les residus 11 a 30 de l'extension n-terminale de l'asprs de rat, susceptible de former une helice amphiphile, est un des elements cles de cette association

bioRxiv (Cold Spring Harbor Laboratory), Dec 4, 2023

Angewandte Chemie International Edition

Preventing the misfolding or aggregation of transactive response DNA binding protein with 43 kDa ... more Preventing the misfolding or aggregation of transactive response DNA binding protein with 43 kDa (TDP‐43) is the most actively pursued disease‐modifying strategy to treat amyotrophic lateral sclerosis and other neurodegenerative diseases. In this work, we provide proof of concept that native state stabilization of TDP‐43 is a viable and effective strategy for treating TDP‐43 proteinopathies. Firstly, we leveraged the Cryo‐EM structures of TDP‐43 fibrils to design C‐terminal substitutions that disrupt TDP‐43 aggregation. Secondly, we showed that these substitutions (S333D/S342D) stabilize monomeric TDP‐43 without altering its physiological properties. Thirdly, we demonstrated that binding native oligonucleotide ligands stabilized monomeric TDP‐43 and prevented its fibrillization and phase separation in the absence of direct binding to the aggregation‐prone C‐terminal domain. Fourthly, we showed that the monomeric TDP‐43 variant could be induced to aggregate in a controlled manner, wh...

Nature Communications, Sep 21, 2021

The EMBO Journal, Sep 17, 2009

Biochemical Pharmacology, Nov 1, 2011

Aberrant and constitutive NF-kB activation are frequently reported in numerous tumor types, makin... more Aberrant and constitutive NF-kB activation are frequently reported in numerous tumor types, making its inhibition an attractive target for the treatment of certain cancers. NEMO (NF-kB essential modulator) is the crucial component of the canonical NF-kB pathway that mediates IkB 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-kB pathway at the IKK level in different cell types stimulated by a variety of NF-kB signals. Circular dichroïsm and fluorescence studies showed that UBI exhibits an increased a-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-kB signaling and would provide a new tool for studying the complex role of NF-kB in inflammation and cancer.

bioRxiv (Cold Spring Harbor Laboratory), Dec 23, 2022

Biochemistry, 1995

Conformational studies were performed on the synthetic tricosapeptide N-acetyl-SKKALKKLQKEQEKQRKK... more Conformational studies were performed on the synthetic tricosapeptide N-acetyl-SKKALKKLQKEQEKQRKKEER-amide, representing the highly basic segment (residues 30-52) of the N-terminal extension of yeast cytoplasmic aspartyl-tRNA synthetase. Circular dichroism experiments show that, in aqueous solution at neutral pH, the peptide adopts a random conformation. The effects of pH, temperature, addition of trifluoroethanol (TFE), and titration with polyanions on the conformation of the peptide were studied. In TFE or in the presence of an equimolar concentration of (phosphate)ls, the peptide adopts a 100% a-helical conformation. A partially a-helical conformation is induced by (phosphatek or d(pT)g (respectively 40% and 35% helical content). Raising the pH in aqueous solution promotes 75% a-helicity, with a transition pK of 9.9 reflecting deprotonation of lysine residues. On the basis of these results, nuclear magnetic resonance studies were carried out in TFE as well as in aqueous solution in the presence of (phosphate)lg, to determine the structure of the molecule. Complete 'H resonance assignments were obtained by conventional two-dimensional NMR techniques. A total of 138 interproton constraints derived from NOESY experiments were used to calculate the three-dimensional structure by a two-stage distance geometryhimulated annealing procedure. The two deduced structures were highly similar and show that nine cationic residues are segregated on one face of a helical structure, providing an ideal polycationic interface for binding to polyanionic surfaces. Compared to its prokaryotic homologue, yeast cytoplasmic aspartyl-tRNA synthetase carries a cationic N-terminal extension of about 100 amino acid residues (Sellami et al., 1986; Eriani et al., 1990). Similar positively charged N-terminal extensions of varying length and sequence were found in other yeast aminoacyl-tRNA synthetases, including valyl-, threonyl-, and lysyl-tRNA synthetases [reviewed in Mirande (1991)l. It was shown previously that lower eukaryotic aminoacyl-tRNA synthetases generally display much higher affinity for polyanionic chromatographic supports than do the corresponding enzymes from prokaryotes (Cirakoglu & Waller, 1985). The finding that excision of the lysine-rich N-terminal extensions from yeast lysyl-(Cirakoglu & Waller, 1985) and aspartyl-tRNA synthetases (Lorber et al., 1988; Eriani et al., 1991) generates modified enzymes that retain catalytic activity yet display much reduced affinity for immobilized heparin supported the view that the high affinity toward polyanionic carriers was mainly conferred by their cationic N-terminal extensions. In line with this conclusion, it was recently shown that whereas yeast valyl-and lysyl-tRNA synthetases bind strongly to micro-+ Supported by grants from the Centre National de la Recherche Scientifique, from the Ligue Nationale contre le Cancer, and from the Association pour la Recherche contre le Cancer.

Journal of Biological Chemistry, Jul 1, 1999

Journal of Biological Chemistry, Dec 1, 2004

NF-B essential modulator/IKK-␥ (NEMO/IKK-␥) plays a key role in the activation of the NF-B pathwa... more NF-B essential modulator/IKK-␥ (NEMO/IKK-␥) plays a key role in the activation of the NF-B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (