Enzymatic Regulation of Cellular Activities - CNRS URA 2185  


  HEADProf. VERON Michel / michel.veron@pasteur.fr
  MEMBERSNEMO Group Permanents : Dr AGOU Fabrice, Chef de Laboratoire IP Dr FONTAN Elisabeth, Chargée de Recherche IP CHIARAVALLI Jeanne, Ingénieur de Recherche IP TRAINCARD François, Ingénieur de Recherche IP Post-doctorant Dr KAMINSKA Monika, PhD Stagiaire pré-doctorante : Ngadjeua Flora
Pharmacology Group Permanents : Dr ROUGEOT Catherine, Chef de Labotoire IP DUFOUR Evelyne, Technnicienne Supérieure IP Stagiaire pré-doctorante : BOGEAS Alexandra
Secrétaire : TRAN Catherine
Agent de laboratoire : KACER Martine


  Annual Report

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

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

The activation of the NF-kB pathway, initially considered as mainly involved in inflammation and immune responses, is now recognized as playing a crucial role in apoptosis and cancer. 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 cultured cell lines.

The 419 amino-acids NEMO polypeptide is composed of several domains including an oligomerization domain, comprised within the C terminal half of the protein which is formed of two coiled-coils (CC2 and LZ) and a Zinc finger at the C-terminus of the polypeptide. The solution structure of the Zinc finger was solved by NMR showing that it constitutes a functional ubiquitin binding domain and a structural model of the complex with mono-ubiquitin was proposed on the basis of mutagenesis and NMR (Collab. Unité de RMN des biomolécules).

The formation of oligomeric complexes of NEMO polypeptide through the interaction of its CC2-LZ coiled-coils is necessary for biological activity. NEMO also binds polyubiquitin chains through a sub-region of the CC2-LZ domain called NOA which is conserved in 5 proteins (NEMO, Optineurin [also called NRP] and Optineurin 1, 2 and 3). While K48- or linear polyubiquitin chains also bind to NOA, binding of K63 linked polyubiquitin chains, which is necessary for NEMO activity, specifically occurs through a bipartite site involving both the NOA and the Zn finger domains (Collab. Unité de Signalisation moléculaire et activation cellulaire).

The crystal structure of the oligomerization/polyubiquitin binding domain of NEMO was determined at 2.9 A resolution in the presence of design ankyrin repeats proteins (DARpins) which helped its crystallisation. The CC2-LZ domain is formed of coiled-coils forming a dimer with an unusual stammer at their junction. Physico-chemical techniques were used with several structure based mutants altered in their ability to activate the NF-kB pathway and in mono- or poly-ubiquitin binding, to investigate the coupling between ubiquitin binding and dimerization.

Mutations in NEMO coding sequence have been identified in patients with rare human genetic diseases, leading to immuno-deficiency (ID) with or without anhidrotic ectodermal dysplasia (EDA-ID). We studied the physico-chemical properties of mutations in the CC2-LZ or in the Zn finger domain and correlated them to dimeric assembly and to poly-ubiquitin binding (collab. Prof. J L Casanova, Hôpital Necker, Paris).

We are also developping an R&D approach to find inhibitors of NEMO function which might stimulate apoptosis in tumoral cells through an inhibition of the NF-kB pathway (in collaboration with a french Pharma company).

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

The enkephalins are mammal opioid neuropeptides,which regulate spinal and central processing of pain sensation, as well as emotional and motivational behaviors.Their action is specifically transmitted viaµ- andδ-opioid membrane receptors. However in vivo, circulating enkephalins are rapidly inactivated by the concomitant action of two membrane-bound metallo-ectopeptidases, which are co-located with opioid receptors, namely, neutral endopeptidase (NEP) and aminopeptidase-N (AP-N).

In a multi-level approach, using genetic, biochemical, molecular and behavioral pharmacology methodologies, we discovered the existence of physiological dual inhibitors of both enkephalin-inactivating Zn-ectopeptidases, NEP and AP-N in mammals. We named one of these compounds rat Sialorphin. The corresponding QHNPR-pentapeptide, was identified using an integrative post-genomic approach. Human Opiorphin, the QRFSR-pentapeptide, was subsequently discovered using a functional biochemical approach.The discovery of such novel endogenous upstream regulators in human, is of crucial interest from physiological and therapeutic points of view. Indeed, endogenous human Opiorphin appears to intervene in the process of adaptation mediated by enkephalins, which are associated with pain and emotion, and particularly in the regulation of the pronociception-antinociception and depression-motivation homeostatic equilibrium.

Using molecular and cellular pharmacology approaches, we demonstrated that Opiorphin is a dual inhibitor of both human NEP and AP-N ectopeptidases, and that it protects Met-enkephalin from degradation by these two ecto-peptidases. In vivo, Opiorphin displays potent analgesic activity in chemical and mechanical pain rat models by activating endogenous enkephalin-related opioid-dependent transmission. Its pain-suppressive potency and dose-effect is comparable to morphine.

Structure/Activity relation studies and chemical optimization of Opiorphin native peptide allowed to generate functional derivatives endowed with better bioavailability properties (lipophilicity and metabolism resistance) than the native peptide. These functional peptidomimetics of human Opiorphin would lead to potent analgesics in terms of dose/effect, delay and time duration of action in various morphine-sensitive pain modelsand could lead to new anti-pain drug-candidates.

We are also developing biomedical projects to provide the currently missing knowledge on this new mediator of opioid pathways in human. Thus, to explore the profile of secretion and distribution of Opiorphin in healthy human, a clinical research protocol was established. This project in physiological situation, will build-up bases for a further approach to identify human pathological states up-regulating or down-regulating the levels of circulating Opiorphin.

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



  Publications

Laplantine, E., Fontan, E., Chiaravalli, J., Lopez, T., Lakisic, G., Véron, M., Agou, F. and Israël, A. (2009) NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain. EMBO J., 28, 2885-2895.

Cordier, F., Grubisha, O., Traincard, F., Véron, M., Delepierre, M. and Agou, F. (2009) The Zinc Finger of NEMO is a functional ubiquitin-binding domain. J. Biol. Chem. : 284, 2902-2907.

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.

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.

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.



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Activity Reports 2009 - Institut Pasteur
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