Enzymatic Regulation of Cellular Activities - CNRS URA 2185  


  HEADProf. VERON Michel (DR1-CNRS) / michel.veron@pasteur.fr
  MEMBERSDr AGOU Fabrice, Chef de Laboratoire IP / Dr FONTAN Elisabeth, Chargée de Recherche IP / Dr FSIHI Hafida, Chargée de Recherche IP / Dr ROUGEOT Catherine, Chef de Laboratoire IP
Dr GRUBISHA Olivera, PhD / Dr KAMINSKA Monika, PhD
FOLKIERSKA-ZUKOWSKA Monika / CHIARAVALLI Jeanne, Ingénieur de Recherche IP / TRAINCARD François, Ingénieur de Recherche IP
DUFOUR Evelyne, Technnicienne Supérieure IP / TRAN Catherine / AYCIN Pinar


  Annual Report

The Laboratory is organized around two different projects, each under the responsibility of senior investigators.

1. NEMO, an essential regulator of the NF-kB signal transduction pathway (PI, F. Agou).

The activation of the NF-kB pathway is a hallmark of inflammation and immune responses. Originally considered as mainly involved in inflammation, its role in apoptosis and cancer is now well established. Thus, inhibitors of the pathway are potential pro-apoptotic anti-cancerous agents. A central regulation of the pathway relies upon the IKK complex which comprises two protein kinases and the scaffolding protein NEMO (NF-kB Essential MOdulator). We study NEMO structure and function by combining structural and biochemical approaches in vitro, and cell biology techniques using cell lines.

Formation of multi-subunit complexes of the 419 amino-acids NEMO polypeptide is necessary for its biological activity. The minimum oligomerization domain, comprised within the C terminal half of the protein, is formed of 2 coiled-coils named CC2 and LZ. Synthetic peptides mimicking the LZ coiled-coil have specific inhibitory effects (IC50in the M range) on both the oligomerization of NEMO in vitro and the activation of the pathway in cultured cells (Collab. F. Baleux, Unité de Chimie Organique). One of these peptides also induces apoptosis of tumoral cells isolated from patients with myelodysplactic syndromes and acute myeloïd leukemia. Thus, the isolation of NEMO oligomerization inhibitors is relevant to the search for reagents that could specifically stimulate apoptosis of cancer cells. In this background, we are developping a R&D program in collaboration with a french Pharma company.

Mutations in NEMO coding sequence were identified in patients with rare human genetical diseases, leading to immuno-deficiency (ID) with or without sign of anhidrotic ectodermal dysplasia (EDA-ID). Our approach is to study the biochemical properties of the corresponding recombinant proteins. We determined the structure of the Zinc finger domain at the C-terminus of NEMO by NMR. We identified a putative ubiquitin binding site in this domain and a structural model of the complex with mono-ubiquitin was proposed on the basis of mutagenesis and NMR (Collab. F. Cordier, Unité de RMN des biomolécules). We are currently investigating the structural and functional effects of several new disease-associated mutations recently identified in this domain as well as in the CC2-LZ domain in the laboratory of Prof. J L Casanova (Hôpital Necker, Paris).

The CC2-LZ domain was co-crystallized with proteins from the ankyrin family which were selected by “ribosome display”. The structure of the complex was solved at 2.9 A (Collab. S. Duquerroy, Unité de Virologie structurale). We are now investigating the contribution of K63 poly-ubiquitin binding to the C-terminal domain of NEMO for the activation of the IKK complex (Collab. E. Laplantine, Unité de Signalisation Moléculaire et Activation Cellulaire).

2. Rat Sialorphin and human Opiorphin, new modulators of opioïd-dependent pathways (PI, C. Rougeot)

The enkephalins are mammal opioïd neuropeptides,which regulate spinal and central processing of analgesic mechanisms, as well as emotional and motivational behaviors.Their action is specifically transmitted, like for morphine, viaµ- andδ-opioïd membrane receptors. However in vivo, circulating enkephalins are rapidly inactivated by two metallo-ectopeptidases, NEP (Neutral-EndoPeptidase) and AP-N (AminoPeptidase-N).

The combination of genetic, biochemistry, molecular and behavioral pharmacology approaches allowed to discover for the first time the existence of physiological inhibitors of two enkephalin-inactivating Zn-ectopeptidases in mammals, Sialorphin (QHNPR) in Rat and, more recently, Opiorphin (QRFSR) in Humans.

The functional characterization of Sialorphin was based on the molecular identification of membrane receptor-sites that bind circulating Sialorphin, in vivo. We determined its functional specificity in vivo as a physiological ligand and a competitive inhibitor of rat NEP. In parallel, we showed that it is a potent inhibitor of painful sensation and a physiological modulator of environmental and socio-sexual motivation in male rats using behavorial models (Collab. M. Messaoudi, Preclinical research centre , Vandoeuvre-lès-Nancy).

A functional biochemical approach allowed us to discover a functional analog of Sialorphin in humans, which wenamed Opiorphin. Using molecular and cellular pharmacology approaches, we demonstrated that Opiorphin is a dual inhibitor of both hNEP and hAP-N ectopeptidases, and that it protects Met-enkephalin from degradation by these two ecto-enzymes. In vivo, Opiorphin displays potent analgesic activity in chemical and mechanical pain rat models by activating endogenous opioïd-dependent transmission. Its pain-suppressive effect in these models is comparable to morphine.

Given the functions mediated by the endogenous opioïdergic pathways, the discovery Opiorphin is of crucial importance from a physiological and physio-pathological point of view. Indeed, human Opiorphin could be involved in the process of enkephalins-mediated adaptation which are associated with pain and emotion, and in particular in the regulation of the homeostatic equilibrium pronociception-antinociception and depression-motivation. Our main objective aims to understand the mechanisms of synthesis and secretion of this new mediator of opioïd pathways in human. Opiorphin may also have important therapeutic implications and we are currently investigating the effects of conformationally restricted Opiorphin mimetics with the appropriate pharmacokinetic and biodistribution properties needed for putative new anti-pain drug-candidates.

Keywords: NF-kB pathway ; IKK complex ; NEMO ; Protein structure; Bioactive peptides ; Opioïd pathways ; Pharmacology; Cancer & inflammation; Pain



  Publications

Cordier, F., Vinolo, E., Véron, M., Delepierre, M. and Agou, F. (2008) Solution structure of NEMO Zinc Finger and impact of an anhidrotic Ectodermal Dysplasia with immunodeficiency-related point mutation. J. Mol. Biol. : 377, 1419-1432.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18313693

Davies, K. P., Tar, M., Rougeot, C. & Melman, A. (2007). Sialorphin (the mature peptide product of Vcsa1) relaxes corporal smooth muscle tissue and increases erectile function in the ageing rat. British J. Urol. Int. 99, 431-435.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17026587

Fontan, E., Traincard, F., Lévy, S. G., Yamaoka, S., Véron, M. and Agou, F. (2007) NEMO oligomerization in the dynamic assembly of the IkappaB kinase complex. FEBS J., 274, 2540-2551.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17419723

Vinolo, E., Sebban, H., Chaffotte, A., Israël, A., Courtois, G., Véron M. and Agou, F. (2006) 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. J. Biol. Chem., 281, 6334-6348.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16379012

Wisner, A., Dufour, E., Messaoudi, M., Nejdi, A., Marcel, A., Ungeheuer, M. N. & Rougeot, C. (2006). Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways. Proc. Natl. Acad. Sci. U S A 103, 17979-17984.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17101991



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