|Biochemistry of Macromolecular Interactions - CNRS URA2185|
|HEAD||Dr Daniel LADANT / firstname.lastname@example.org|
|MEMBERS||Office staff: LENOIR Lucile, Institut Pasteur, email@example.com, BENEDIC Marie-Bénédicte, Institut Pasteur, firstname.lastname@example.org
Researchers : CHENAL, Alexandre, Institut Pasteur, Chargé de Recherche, email@example.com, KARIMOVA Gouzel, Institut Pasteur, Chargée de Recherche, firstname.lastname@example.org, LADANT Daniel, CNRS, DR2, email@example.com
Scientific trainees : ROBICHON Carine, Postdoctoral fellow, firstname.lastname@example.org, SPIACZKA-KARST Johanna, Postdoctoral fellow, email@example.com, ULLMANN Agnès, DREM CNRS, firstname.lastname@example.org, WOZNIAK Anna, Postdoctoral fellow, email@example.com
Other personnel : DAVI Marilyne, Institut Pasteur, technician, firstname.lastname@example.org
Our research interests are mainly focused on the study of the molecular mechanisms that underlie protein-protein and protein-membrane interactions, using as a model system a bacterial toxin, the adenylate cyclase (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough. We are currently pursuing two major themes of research:
I CyaA toxin: Molecular mechanisms of action and application in vaccinologyCyaA is able to invade eukaryotic cells where it is activated by calmodulin to produce supra physiological levels of cAMP. CyaA is a 1706 residue-long protein with a unique mechanism of penetration into eukaryotic cells: after binding through its C-terminal part to a receptor, the αMβ2integrin, at the surface of target cells, the central region is inserted into the plasma membrane and, then, the N-terminal catalytic domain is directly translocated across the membrane into the cytosol of the cells. How CyaA delivers its catalytic domain across the cell membrane remains largely unknown. To address this question, we are exploring the biophysical properties of the CyaA protein in solution or in association with membranes, by using a combination of biochemical and spectroscopic approaches. In 2007, our studies were focused primarily on the structural, hydrodynamic and thermodynamic properties of two isolated CyaA domains, the N-terminal catalytic domain and the C-terminal moiety that is involved in the interaction with the integrin receptor and in the binding of calcium, an essential co-factor for toxin entry into target cells. The effects of ligand binding (eg, calcium and/or calmodulin) on these domains have been characterized.
Another major point of interest in CyaA is that this protein is an efficient vaccine vehicle as shown previously, in collaboration with Claude Leclerc and her team: antigens can be genetically grafted into CyaA and delivered in vivoto dendritic cells to elicit specific T cell responses. In 2007,recombinant CyaA proteins carrying the HIV Tat antigen have been produced and their immunological properties are currently evaluated in C. leclerc’s laboratory.
II In vivoanalysis of protein-protein interactions.
Our second research theme is the study of the assembly of multi-molecular membrane-associated protein complexes in vivo, in particular by exploiting a CyaA-based two-hybrid screening technology (BACTH), previously designed in our group. This genetic assay is based on the functional complementation between two CyaA fragments to restore a cAMP-signaling cascade in an Escherichia coli cyastrain (figure 1). Our current work is focused on the assembly of the cell division machinery of E. coli, by combining in vivointeraction assays (using BACTH system) with genetic approaches and in situprotein localization (using GFP fusions). We have characterized several polypeptides that were isolated by two-hybrid screening as partners of the cell division protein FtsL. These polypeptides might be novel component of the cell division apparatus and could play a role as adaptor proteins to modulate septum formation. Besides, we are also studying the molecular basis of the interaction between 3 components FtsQ, FtsL, and FtsB, that form an essential sub-complex of the cell division machinery.
Keywords: toxin, adenylate cyclase, vectorisation, Two-hybrid, cell division, vaccinology, biotechnology
S. Vougier, J. Mary, N. Dautin, J. Vinh, B. Friguet, D. Ladant (2004) Essential role of methionine residues in calmodulin binding to Bordetella pertussis adenylate cyclase, as probed by selective oxidation and repair by the peptide methionine sulfoxide reductases. J Biol Chem. 279: 30210-30218 (PMID: 15148319)
G. Karimova, N. Dautin, and D. Ladant (2005) Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis. J. Bact.. 187:2233-2243 (PMID: 15774864)
C. Bauche, A. Chenal, O. Knapp, C. Bodenreider, R. Benz, A. Chaffotte, and D. Ladant (2006) Structural and functional characterization of an essential RTX sub-domain of Bordetella pertussis adenylate cyclase toxin. J Biol Chem. 281 :16914-16926 (PMID: 16627468)
F. Dal Molin, F. Tonello, D. Ladant, I. Zornetta, I. Zamparo, G. Di Benedetto, M. Zaccolo, and C. Montecucco (2006) Cell entry and cAMP imaging of anthrax edema toxin. EMBO. J. 25:5405-5413 (PMID: 17082768)
P. Berraondo, C. Nouzé, X. Préville, D. Ladant, C. Leclerc(2007) Eradication of Large Tumors in Mice by a Tritherapy Targeting the Innate, Adaptive, and Regulatory Components of the Immune System. Cancer Research67:8847-8855 (PMID: 17875726)
Activity Reports 2007 - Institut Pasteur
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