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  Director : SUSIN, Santos A. (susin@pasteur.fr)


  abstract

 

The apoptosis and Immune System team, who started their work in March 2002, is doing research on the mechanisms of caspase-independent cell death in the immune system. The specific aims of our scientific project are to: 1) define the biochemical mechanisms of caspase-independent cell death induced by CD47 activation in B-chronic lymphocytic leukemic (B-CLL) cells; 2) identify the molecular mechanisms of one of the main agents implicated in caspase-independent apoptosis, the mitochondrial protein AIF; 3) evaluate the contribution of this protein to the execution of cell death.



  report

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I. Biochemical mechanisms of caspase-independent cell death mediated by CD47 in B-chronic lymphocytic leukemic (B-CLL) cells (Gaël Roué).

During apoptosis (programmed cell death), cells set up a "suicide mechanism" that gives rise to numerous changes. The most significant changes include cell shrinkage, disruption of the mitochondrial transmembrane potential, DNA fragmentation and phosphatidylserine externalization. Thanks to these hallmarks, the apoptotic cell can be recognized and eliminated. In mammals, programmed cell death is closely linked to the activation of a set of proteinases, called caspases. Although caspases are necessary for the execution of apoptosis in many systems, they are not always required. In this case, the process is called caspase-independant cell death.

The involvement of the antigen CD47 (IAP) via its physiological ligand, thrombospondin, or via an anti-CD47 agonist antibody, that mimics the effects of the ligand, induces leukocyte adhesion, migration, phagocytosis, and cell motility through its association with integrins of b1, b2, and b3 families. It also induces a form of caspase-independent cell death, characterized by cytoplasmic events such as the externalization of phosphatydilserines and the fall in mitochondrial transmembrane potential. Death induced by an anti-CD47 antibody seems independent of signaling pathways described in classical apoptosis. Indeed, dying cells display intact or slightly modified nuclei (absence of oligonucleosomal fragmentation). Cytoskeleton rearrangement —linked to the Cdc42/WAS protein signalling pathway-, and K+ efflux are also involved in this new form of planned cell death, but the biochemical mechanisms and the proteins implicated in CD47-dependent apoptosis still have to be elucidated. In the context of B-cell chronic lymphocytic leukaemia (the most common hematological malignancy in Western countries, that makes up nearly 30 % of all leukaemias), the understanding of the mechanisms that regulate caspase-independent apoptosis could contribute to the development of new therapeutic means, aimied at increasing a stimulation of the anti-tumour response among patients. Indeed, except for a few isolated cases of spontaneous remission, the immunological anti-tumour response does not seem efficient among most patients, and we do not observe any regression of the tumour mass, even among patients presenting a slow evolution of their disease. Recent studies, made by Dr Sarfati's team (who collaborate on this project), show that B-lymphocytes from B-CLL are more sensitive to death provoked by CD47 than B and T lymphocytes from healthy donors. These elements lead us to think that understanding the death mechanisms involved in CD47 signalling will permit the development of a treatment for B-CLL. The combination of 1) a thorough knowledge of the biochemical and molecular mechanisms involved in this kind of cell death, 2) the achievement of the induction of an anti-tumour response in vitro and 3) the use of new strategies aimied at increasing dendritic cells in vivo, could result in the development of new therapeutic means for B-CLL.

II. Molecular mechanisms determining the function of AIF (Cécile Delettre-Cribaillet & Victor J. Yuste-Mateos).

One of the main agents of caspase-independent apoptosis is AIF (or "Apoptosis Inducing Factor"), characterized by Susin et al. in 1999 (Nature, vol 397, pp. 441-446). AIF is a mitochondrial intermembrane flavoprotein, that fulfills both oxydoreductase and apoptotic functions. Following an apoptotic stimulus, AIF translocates from mitochondria to the nucleus where it induces two specific signals of caspase-independent death: peripheral chromatin condensation and large-scale (50Kbp) DNA fragmentation. AIF by itself is not able to provoke DNA fragmentation, which means that the process is accomplished through cooperation between AIF and other unknown factor(s). In addition to its effects at the nuclear level, AIF also has an effect on the isolated mitochondria. Indeed, in the presence of other unidentified cytosolic protein(s) it provokes mitochondrial swelling, and the loss of the mitochondrial transmembrane potential. Its ectopic expression in the cytosol also causes phosphatidylserines externalization, according to a mechanism that still has to be determined. All these results enable us to make several important reflections on the mechanism involved in AIF-induced caspase-independent cell death and on the identification of proteins implicated in the AIF-mediated apoptosis pathway.

The aim of our project is to characterize the cellular and molecular mechanisms that determine AIF's function, in order to fill a gap in our present knowledge of AIF, as well as its modes of action. To achieve this, we wish to answer several questions: 1) What are the cytosolic, nuclear and mitochondrial factors that are modulated by AIF, or that interact with AIF? Our goal is to achieve (in collaboration with the "Plate-forme Protéomique") an identification of nuclear, cytosolic and mitochondrial partners of AIF. 2) What is the structure/function relationship of AIF? The C-terminal part of AIF shows polar and hydrophobic areas, as well as reactive residues, suggesting a possible C-terminal localization for the apoptotic activity of AIF. The N-terminal domain has a putative site of interaction between AIF and its cofactor, FAD, and a site of interaction with the donor of electrons of the protein, NADH. It would thus be interesting to mutate the different residues that belong to these functional areas of AIF, in order to determine which ones are important in each of the two functions of the protein. 3) There are two splice variants of AIF. What is the involvement of each splice variant in cell death execution? To verify their apoptotic roles, we will use RNA interference (RNAi) to eliminate, individually, their expression in cells. It would be important to characterize their distribution and their particular involvement in the execution of cell death in normal and pathological conditions.

Taken together, these experiments should elucidate the role of AIF in the regulation of caspase-independent cell-death. The comprehension of the mechanism of AIF function should provide conceptual advances for therapeutic interventions on deficient or excessive caspase-independent apoptosis.

III. Development of new tools for the intracellular transfer of biomolecules (Victor J. Yuste-Mateos & Nadine Robert, in collaboration with Frédéric Dessauge & Alphonse Garcia, of the Laboratory of Signalisation Immunoparasitaire at the Pasteur Institute).

This work, performed in collaboration with the Laboratory of Signalisation Immunoparasitaire, consists of testing the biological effect (intracellular penetration and effect on survival) of a series of peptides (coming from a panel of different proteins) whose sequences imitate the sites of interaction with the proteins of the protein phosphatase 2A (PP2A) family. We have identified several peptides, derived from three proteins (CK2a, Vpr-HIV-1, and protamine), that are able to interact with PP2A. From these peptides, we have characterized, on the one hand, penetrating peptides with no biological effect that would be able to transfer macromolecules into the cytoplasm or in the nucleus of Jurkat/HeLa cells, and, on the other hand, penetrating peptides able to induce apoptosis in these cells. The apoptosis induction mechanisms and the potential anti-tumour properties of these peptides are currently being examined.

Keywords: Apoptosis, Apoptosis Inducing Factor (AIF), Cancer, B-CLL, Caspase-independent cell death



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
    SUSIN, Santos A., CNRS, Senior Scientist,susin@pasteur.fr DELETTRE-CRIBAILLET, Cécile, Postdoc

ROUÉ, Gaël, Postdoc

YUSTE-MATEOS, Victor J., Postdoc

ROBERT, Nadine (Technician,nadou@pasteur.fr)

Activity Reports 2002 - Institut Pasteur
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