Activation de la caspase-8 dans des contextes apoptoiques et non apoptotiques: Caractérisation de nouvelles voies d'activation et des partenaires impliqués – caspase-8

Apoptosis, the main type of programmed cell death, is involved in controlling embryonic and fetal development, in tissue renewal, and in establishing and regulating the immune system. Apoptosis is a basic mechanism for controlling cellular activation and plays a critical role in maintaining cellular homeostasis. Many studies have highlighted the importance of caspase-family proteases in apoptosis during the last few years. Among these, caspase-8 plays a critical role as it is involved in the control of both apoptosis and proliferation. Apical caspase-8 is centrally involved in initiating apoptotic pathways promoted by various stimuli, including death receptors of the TNF-R family, e.g. Fas. Caspase-8 activation is dependent on its association with the FADD protein in this context. We recently reported that human B lymphocytes stimulated through the BCR, TGF-ß or heavy metals can also activate caspase-8. However, this occurs through a novel pathway that is independent of the DED of FADD. Unlike the FADD-dependent pathway, this new caspase-8 activation pathway is controlled by transduction pathways involving MAPK family kinases. Caspase-8 was found to be critical for differentiation and/or proliferation of B and T lymphocytes, and to be involved in differentiation of hematopoietic progenitors and cells of the myelomonocytic lineage. Thus, caspase-8 plays a central role in controlling immune system homeostasis. This role does not always depend on its apoptotic properties. Therefore, it is important to elucidate the nature of these novel caspase-8 activation pathways and to characterize new molecules that activate or are substrates of caspase-8 in apoptotic or nonapoptotic contexts. Caspase function may also depend on its cellular location and thus on the nature of the substrates in these cellular compartments. Although most caspase-8 is present in the cytoplasm and interacts indirectly with transmembrane receptors, we recently reported that there are sumoylated nuclear forms of caspase-8. We intend to continue this study by characterizing and investigating the function of nuclear partners of sumoylated caspase-8. We will identify these new caspase-8 partners by two methodological approaches: yeast two-hybrid screening and mass spectrometry analysis. The yeast two-hybrid approach has the advantage of being able to characterize novel partners of bait protein from cDNA libraries. However, this method is limited by the need to use a cDNA library from a given cell type. This skews the diversity of cDNA present in the cell during formation of the library. We propose to overcome this problem by using mass spectrometry to characterize new caspase-8 partners directly from cell lysates of various cell types at various stages of activation. Simultaneous use of these two approaches will enable us to characterize new partners upstream from caspase-8 activation (regulator molecules) and downstream from this caspase (substrates). Previous two-hybrid screening by our group indicated that caspase-8 interacts with the protein SUMO-1. This sumoylation of caspase-8 (confirmed by mass spectrometry) controls its nuclear location. We will use two-hybrid screens in collaboration with the French company "Hybrigenics" to search for new molecules that can interact with caspase-8. This company has developed a new generation of screens that are broader and more informative than traditional screens. Mass spectrometry will allow us to characterize caspase-8 partners directly from cell lysates and nuclear fractions of a given cell type at a selected stage of activation. Caspase-8-associated proteins of interest will be separated by electrophoresis and analyzed by mass spectrometry in our institute (IFR89). This dual approach should allow us to better understand the role of caspase-8 in novel pathways controlling cellular activation and to undertake functional studies needed to determine the biologic relevance of caspase-8 nuclear localization. The use of lentiviral-mediated RNA interference specific for these new partners of caspase-8, either in vitro or in vivo using immunodeficient NOD/SCID mice, will allow us to determine their role in caspase-8 activation in apoptotic and nonapoptotic contexts and to precise the role of caspase-8 during human hematopoiesis. This study will characterize novel caspase-8-associated molecules involved in regulation of the balance between apoptosis and proliferation. Dysfunction of this control causes disruption of cell homeostasis observed in many diseases.