Cellular and Structural Biochemistry - CNRS URA 2185  


  HEADProf. VERON Michel / michel.veron@pasteur.fr
  MEMBERSNEMO Group : 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), Dr DUBOSCLARD Virginie (Post-doctorante), NGADJEUA Flora (doctorante), HENRIQUES Ricardo (doctorant)
Opiorphin Group : Dr ROUGEOT Catherine (Chef de Labotoire IP), DUFOUR Evelyne (Technnicienne Supérieure IP) CHEKHAB Mourad (Master I) TRAN Catherine (Secrétaire)
KACER Martine (Agent de laboratoire)


  Annual Report

We study the relation between structure and function in regulatory proteins and biologically active peptides. The laboratory is composed of two groups. The NEMO Groupstudies an important regulatory protein of the NF-kB signal transdution pathway, using the methods of protein biochemistry, physicochemistry and cell biology, in collaboration with other Unités and technological Platforms on campus in structural biology (X-ray crystallography, NMR and molecular modeling). The Opiorphin group, studies the pharmacokinetics and pharmacodynamic properties of a human peptide regulator which has strong analgesic effects in rats, using methods of peptide biochemistry (FRET-based enzyme assays; RP-HPLC and competitive ELISA) and behavioral pharmacology. It collaborates off-campus with chemists for designing peptidomimetics and with preclinical research center to characterize the effects of Opiorphin and derivatives on pain perception and depressive-like behaviors using rat models.

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

NF-kB pathway activation, considered to be primarily involved in inflammation and immune responses, is now being recognized for its crucial role in apoptosis protection and cancer. Thus, inhibitors of the pathway hold promise as cancer therapeutics and anti-inflammatory agents. Regulation of the pathway relies on the IKK complex, which comprises two protein kinases and the scaffolding/regulatory protein NEMO (for NF-kB Essential Modulator). We study NEMO structure and function using a combination of structural and biochemical approaches for in vitrostudies,and using cell biology techniques for studies in cultured cell lines.

The NEMO polypeptide consists of 419 amino-acids and is composed of several domains, one of which mediates oligomerization. This domain exists within the C terminal half of the protein and is formed by two coiled-coils (CC2 and LZ) and a zinc finger (ZF) that resides at the C-terminus. The solution structure of the ZF was solved by NMR, and showed that it constitutes a functional ubiquitin binding domain (UBD). A structural model of this domain, complexed with mono-ubiquitin, has been proposed on the basis of mutagenesis and NMR studies (collab. Unité de RMN des Biomolécules).

The association of monomers into homo-dimeric complexesis necessary for its biological activity. This occurs viainteractions between their CC2-LZ coiled-coils. NEMO also binds polyubiquitin chains through a second UBD, located within the CC2-LZ domain. This UBD is also called NOA to denote its presence in five different proteins (NEMO,

The crystal structure of the oligomerization/polyubiquitin binding domain of murine NEMO was determined at 2.95 Å resolution in the presence of designed ankyrin repeat proteins (DARPins) which facilitated crystallization. Recently, the structure of human NEMO was solved at 2.6 Å using new methods of purification and crystallization. The CC2-LZ domain is formed by dimeric coiled-coils, which have an unusual stammer configuration at their junction. To investigate the details of the relationship between ubiquitin binding and dimerization, we performed physico-chemical experiments using mutant proteins that were altered in their ability to either activate the NF-kB pathway or bind ubiquitin.

Mutations in the NEMO coding sequence were identified in patients having rare genetic diseases which lead to immunodeficiency (ID) possibly linked to anhidrotic ectodermal dysplasia (EDA-ID). We studied the physico-chemical properties of several NEMO proteins harboring CC2-LZ- or ZF-domain mutations, which correlated with defects in dimer assembly and/or in polyubiquitin binding (Collab. Prof. J L Casanova, Hôpital Necker, Paris).

We are also developing an R&D approach to find inhibitors of NEMO which might stimulate apoptosis in tumor cells through inhibition of the NF-kB pathway. A new peptide (Ubiquitin . UBI thus provides a specific tool to study the predominant role of K63-linked polyubiquitin chains in the recruitment of NEMO and the IKK complex to the receptosome during the NF-kB pathway activation process.

Theme 2: Study of Opiorphin peptide modulator (PI, Catherine Rougeot)

In mammals, the most potent opioid neuropeptides are the enkephalins,which regulate spinal and central processing of pain sensation, as well as emotional and motivational behaviors.Their activities are specifically transmitted viahigh-affinity interactions with both the mu- and delta-opioid receptors. However, circulating enkephalins are rapidly inactivated in vivoby the concomitant action of two membrane-bound metallo-ectopeptidases: neutral endopeptidase (NEP) and aminopeptidase-N (AP-N), which co-localize with opioid receptors.

Using a multi-level approach, consisting of genetic, molecular and behavioral pharmacology methods, we discovered the existence in mammals of physiological dual-inhibitors of the two enkephalin-inactivating Zn-ectopeptidases, NEP and AP-N. We identified the rat compound as the pentapeptide QHNPR using post-genomic inverse pharmacology and we named it Sialorphin. A human ortholog, Opiorphin, corresponding to the QRFSR pentapeptide was subsequently discovered using a functional biochemical approach.The discovery of such novel endogenous upstream regulators of ecto-enkephalinases in humans, is of crucial physiological and therapeutic interest. Indeed, inhibiting the degradation of circulating enkephalins, which are released in response to stimuli, increases their half-life and is an effective approach to prolonging their bioavailability and to enhancing their physiological actions.

In a variety of rat models, human Opiorphin was found to be both a powerful inhibitor of pain sensation and a modulator of well-being, through activation of endogenous enkephalin-related opioid-dependent pathways.

Structure/activity relationship studies and chemical optimization of the native Opiorphin peptide allowed us to generate functional Opiorphin-derivatives endowed with better bioavailability properties than the native peptide (i.e., lipophilic character and metabolism resistance). These functional peptidomimetics of human Opiorphin hold much promise as a new class of analgesics with potencies similar to morphine, but devoid of its major side effects. A R&D preclinical program is currently underway in order to assess: the preliminary safety of these peptides in acute toxicity tests, their efficacy in various morphine sensitive pain models and their pharmacokinetic properties.

We have also developed biomedical studies to investigate the role of this new mediator of opioid pathways in humans. Thus, we are exploring the profile of secretion and distribution of Opiorphin in healthy human volunteers. These physiological studies have created a new research basis that will allow us to identify human pathological states which involve the up- or down-regulation of circulating levels of Opiorphin.

Keywords: NF-kB pathway, IKK complex, NEMO, Protein structure ,Cancer and inflammation , Apoptose, Enkephalin-related Opioid pathways , Opiorphin, Molecular and behavioral Pharmacology , Bioactive peptides , Pain



  Publications

  • Rougeot, C., Robert, F., Menz, L., Bisson, J.F. and Messaoudi, M. (2010) Systemically active human Opiorphin is a potent yet non-addictive analgesic without drug tolerance effects. J. Physiol. Pharmacol. 61, 483-490

  • Grubisha, O., Kaminska, M., Duquerroy, S., Fontan, E., Cordier, F., Haouz, A., Raynal, B., Chiaravalli, J., Delepierre, M., Israël, A., Véron, M. and Agou, F. (2010) DARPin-assisted crystallography of the CC2-LZ domain of NEMO reveals a coupling between dimerization and ubiquitin-binding. J. Mol. Biol., 395 89-104.

  • Javelot, H., Messaoudi, M., Garnier, S. and Rougeot, C. (2010) Human Opiorphin is a naturally occurring antidepressant acting selectively on enkephalin-dependent delta-opioid pathways. J. Physiol. Pharmacol. 61, 355-362

  • 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.



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Activity Reports 2010 - Institut Pasteur
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