EUROPEAN ANNEXIN HOMEPAGE
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THE EC PROJECT
Update: 15.12.2004
This website was founded as the Homepage of the former European Collaborative Project BIO4CT960083.
Members
of this project were:
The Groningen Group
University of Groningen
Department of Biophysical Chemistry
Nijenborgh 4
Groningen NL-9747 AG, The Netherlands
 		Alain Brisson

now at: Universite Bordeaux 1
16 Avenue Pey Berland, Pessac, France
The London Group
University College London
Department of Physiology
Gower Street
London WC1E 6BT, United Kingdom
 		Stephen E. Moss
The Martinsried Group
Max-Planck-Institut für Biochemie
Abteilung Strukturforschung
Am Klopferspitz 18a
82152 Martinsried, Germany
 		Robert Huber
The Münster Group
University of Münster
Clinical Research Group for Endothelial Cell Biology
Von-Esmarch-Strasse 56
48149 Münster, Germany
 		Volker Gerke
The Orsay Group
Laboratoire pour l'Utilisation du Rayonnement Synchrotron (LURE)
CNRS, MRESIP, CEA
Bâtiment 209D
Centre Universitaire Paris-Sud
91405 Orsay, France
 		Anita Lewit-Bentley
The Paris Group
Unite de Recherche INSERM U 332
Institut Cochin de Genetique Moleculaire (ICGM)
22 rue Mechain, 75014 Paris, France
 		Françoise Russo-Marie

now at: Bionexis, France
The Perugia Group
University of Perugia
Department of Experimental Medicine and Biochemical Sciences
Via del Giochetto, C.P. 81 Succ. 3
06122 Perugia, Italy
 		Rosario Donato
History of the EC Project
30.09.99 Final Scientific Report and Press Release
01.10.96 Start of the European Collaborative Project BIO4CT960083: "Annexin Structure and Function" with 7 members
Scientific Report
30.09.99
Objectives:
1. X-ray structure determination of individual annexin molecules.
2. Characterisation of annexins in solution.
3. Study of interactions between annexins and other proteins.
4. Analysis of the formation of annexin homo- and hetero-complexes.
5. Elucidation of their function by molecular and electrophysiological techniques.
6. Exploitation of our results for medical purposes.
Main Achievements:
1. X-ray structure determination of individual annexin molecules.
Atomic Force Microscopy imaging of annexin V two-dimensional crystals gave new insights into both the formation of these crystals on a membrane surface, and the overall shape of the molecule attached to the membrane. The structures of annexin 24 from Capsicum anuum and of the annexin III mutant (E231A) have been solved. An important methodological advance was achieved in the structure determination of annexin 24 (Ca32) by producing a stable mutant capable of crystallizing. A further achievement was the generation of a library of 81 annexin models and the use of an automated procedure for the molecular replacement method of solving crystal structures of annexins. In order to provide recombinant expression systems and purified wild-type as well as mutant proteins for biochemical and structural analyses, an eucaryotic expression system for annexin I has been established which will allow the production of purified protein suitable for complex reconstitution with its partner S100C.
We continued to investigate techniques that would allow us to generate useful quantities of recombinant annexin VI from the Pichia expression system. Eventually we decided that recovery of annexin VI from this yeast was not feasible and abandoned any further work using this system. However, we have generated GST fusion proteins of both splice forms of annexin VI and these have been expressed at high levels in bacteria.
2. Characterisation of annexins in solution.
The binding of annexin V to calcium and phospholipids has been further studied by spectroscopic methods. Several annexin III and V mutants have been studied in order to analyse the conformational change in domain III. We are coming closer to an understanding of the way annexins interact with membrane surfaces.
3. Study of interactions between annexins and other proteins.
Further studies of the interactions of annexins V and VI with S100A1 and S100B show that annexin VI, but not annexin V, modulates the effects of S100A1 and S100B on intermediate filament assembly. The annexin VI binding site on S100A1 and S100B is not identical to the desmin/GFAP/tubulin/CapZ/p53 site. Proximity of annexins V and VI with S100A1 and S100B in skeletal muscle cells suggests that annexins V and VI might indeed interact with S100A1 and S100B in vivo.
5. Elucidation of annexin function by molecular and electrophysiological techniques.
Denaturation studies of several annexin V mutants point to an allosteric-like effect of the N-terminus on the binding of the protein to membranes. To approach an understanding of the physiological role of annexins we have generated synthetic annexin II peptides which disrupt preformed complexes of this annexin with its partner p11. When introduced into live cells these peptides interfere with Ca2+-regulated exocytosis suggesting a role of the annexin II-p11 complex in linking secretory vesicles to the plasma membrane. We have continuted our investigation into the consequences of targeted disruption of annexin genes in chicken DT40 cells, and disruption of annexin VI in mice. We have shown that loss of annexin V in DT40 cells leads to multiple phenotypic changes that are probably linked to changes in mitochondrial function. Loss of annexin VI in mice leads to abnormalities in fat deposition in middle age. We have continued our investigation on the function of annexin I, using in vitro and in vivo models. Extracellular annexin I carries anti-inflammatory properties mediated by its N-terminal end, while intracellular annexin I is involved in the control of hepatocyte proliferation probably by regulating the activity of cytosolic phospholipase A2.
Major Scientific Breakthroughs:
  • We have observed annexin II-GFP and annexin I-GFP fusion proteins in living cells for the first time. We have discovered phenotypic changes in cells and mice lacking annexins II, V and VI and are begining to understand the underlying mechanisms.
  • We have solved the structure of two S100 - peptide complexes: that of p11 with the annexin II N-terminal sequence and that of S100C with the annexin I N-terminal sequence. The two structures show a common mode of binding of substrates to S100 proteins: the S100-type dimer is essential for the binding, as the peptides interact with a hydrophobic surface formed by the N-terminal helix of one and the C-terminal helix of the other monomer, as well as the variable central loop. The modified EF hand of p11 adopts the conformation of a calcium-loaded EF hand, thus indicating stabilising features of these folds. The AFM study of crystallisation of annexin V on lipid surfaces presents the first application of this new imaging method to the field of annexins and it provides the first demonstration of the formation of protein 2D crystals on solid supports, offering exciting possibilities in applied sciences. More specifically, it provided the ultimate proof that annexin V molecules form spontaneously ordered arrays of trimers upon membrane-binding.
Press Release
30.09.99
Annexins are ubiquitous and very abundant proteins that bind to membranes in the presence of calcium. Their precise physiological function(s) is not well understood yet. We have decided to tackle the problem by combining efforts from groups that come both from different disciplines, and from different countries of Europe. This past year we have been able to describe the atomic structure of complex of one part of an annexin molecule with another, smaller, protein partner. We have solved the structure of an annexin from a plant, which shows slightly different behaviour from mammalian annexins. We have made progress in the understanding of the distribution of some annexins in certain cells, as well as being able to study cells where certain annexins are absent due to genetic engineering. Most importantly, though, the seven groups spread over Europe have been meeting regularly, exchanging students, ideas and results, thus progressing much more efficiently than in isolation.
© 1997-2007 by ah
Version 8: 15.12.04 / Infrequently updated.