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International audience; Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60(v-src) tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60(v-src), we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60(v-src) inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60(v-src) inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60(v-src) tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60(v-src), we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60(v-src) inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60(v-src) inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.

International audience; Fibrillar collagens are involved in the formation of striated fibrils and are present from the first multicel-lular animals, sponges, to humans. Recently, a new evolutionary model for fibrillar collagens has been suggested (Boot-Handford, R. P., Tuckwell, D. S., Plumb, D. A., Farrington Rock, C., and Poulsom, R. (2003) J. Biol. Chem. 278, 31067–31077). In this model, a rare genomic event leads to the formation of the founder vertebrate fibrillar collagen gene prior to the early vertebrate ge-nome duplications and the radiation of the vertebrate fibrillar collagen clades (A, B, and C). Here, we present the modular structure of the fibrillar collagen chains present in different invertebrates from the protostome Anopheles gambiae to the chordate Ciona intestinalis. From their modular structure and the use of a triple helix instead of C-propeptide sequences in phylogenetic analyses, we were able to show that the divergence of A and B clades arose early during evolution because chains related to these clades are present in protos-tomes. Moreover, the event leading to the divergence of B and C clades from a founder gene arose before the appearance of vertebrates; altogether these data contradict the Boot-Handford model. Moreover, they indicate that all the key steps required for the formation of fibrils of variable structure and functionality arose step by step during invertebrate evolution.

International audience; The Bcl-2 family, including antiapoptotic and proapoptotic members, plays key regulating roles in programmed cell death. We report the characterization of a new member of the bcl-2 family, encoded by herpesvirus of turkeys (HVT). The product of this gene shares 80% homology with Nr-13, an apoptosis inhibitor, which is overexpressed in avian cells transformed by the v-src oncogene. This new gene, that we propose to call vnr-13, is the first member of the bcl-2 family to be isolated among alpha-herpesviruses. Results from cells expressing the HVT-vnr-13 gene product show that the encoded protein inhibits apoptosis and also reduces the rate of cellular proliferation. Contrary to all bcl-2 homologues found in gamma-herpesvirus, which are intronless, vnr-13 has the same organization as the cellular nr-13 gene. Hence, the HVT vnr-13 gene may have been acquired from a reverse transcriptase product of an unspliced precursor RNA, or via direct recombination with the host chromosomal DNA.The Bcl-2 family, including antiapoptotic and proapoptotic members, plays key regulating roles in programmed cell death. We report the characterization of a new member of the bcl-2 family, encoded by herpesvirus of turkeys (HVT). The product of this gene shares 80% homology with Nr-13, an apoptosis inhibitor, which is overexpressed in avian cells transformed by the v-src oncogene. This new gene, that we propose to call vnr-13, is the first member of the bcl-2 family to be isolated among alpha-herpesviruses. Results from cells expressing the HVT-vnr-13 gene product show that the encoded protein inhibits apoptosis and also reduces the rate of cellular proliferation. Contrary to all bcl-2 homologues found in gamma-herpesvirus, which are intronless, vnr-13 has the same organization as the cellular nr-13 gene. Hence, the HVT vnr-13 gene may have been acquired from a reverse transcriptase product of an unspliced precursor RNA, or via direct recombination with the host chromosomal DNA.

International audience; Nr-13 is an anti-apoptotic member of the Bcl-2 family previously shown to interact with Bax. The biological significance of this interaction was explored both in yeast and vertebrate cells and revealed that Nr-13 is able to counteract the pro-apoptotic activity of Bax. The Bax-interacting domain has been identified and corresponds to alpha-helices 5 and 6 in Nr-13. Site-directed mutagenesis has revealed that the N-terminal region of Nr-13 is essential for activity and corresponds to a genuine Bcl-2 homology domain (BH4). The modelling of Nr-13, based on its similarity with other Bcl-2 family proteins and energy minimization, suggests the possibility of electrostatic interactions between the two N-terminal-conserved domains BH4 and BH3. Disruption of these interactions severely affects Nr-13 anti-apoptotic activity. Together our results suggest that electrostatic interactions between BH4 and BH3 domains play a role in the control of activity of Nr-13 and a subset of Bcl-2 family members.

International audience; We previously reported that RSV-transformed quail neuroretina cells (QNR-ts68) were highly resistant to apoptosis provoked by serum withdrawal, and that this property was due to v-Src kinase activity. The present study investigates the cytotoxic effect and the functional mechanism of carbazolequinone-mediated cell death in this system. QNR-ts68 cells were subjected to carbazolequinone treatment and both growth inhibition and cell death induction were examined using formazan assays. Cell death mechanism (both apoptosis and necrosis) was confirmed through phosphatidyl serine exposure and propidium iodide incorporation. Furthermore, the effect of active carbazolequinone was inhibited by a pan caspase inhibitor. Cytofluorimetric and immunofluorescence data demonstrated the activation of caspase-3 and the involvement of mitochondria. Therefore, this study clearly indicates that carbazolequinones could induce cell death in transformed cells displaying high levels of antiapoptotic tyrosine kinase activity. Further investigations would be necessary to elucidate the mechanisms by which these carbazolequinones act as antitumor agents.We previously reported that RSV-transformed quail neuroretina cells (QNR-ts68) were highly resistant to apoptosis provoked by serum withdrawal, and that this property was due to v-Src kinase activity. The present study investigates the cytotoxic effect and the functional mechanism of carbazolequinone-mediated cell death in this system. QNR-ts68 cells were subjected to carbazolequinone treatment and both growth inhibition and cell death induction were examined using formazan assays. Cell death mechanism (both apoptosis and necrosis) was confirmed through phosphatidyl serine exposure and propidium iodide incorporation. Furthermore, the effect of active carbazolequinone was inhibited by a pan caspase inhibitor. Cytofluorimetric and immunofluorescence data demonstrated the activation of caspase-3 and the involvement of mitochondria. Therefore, this study clearly indicates that carbazolequinones could induce cell death in transformed cells displaying high levels of antiapoptotic tyrosine kinase activity. Further investigations would be necessary to elucidate the mechanisms by which these carbazolequinones act as antitumor agents.

International audience; In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans. Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes. The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2. RT-PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys. Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end. Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized. Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope. Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans. Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes. The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2. RT-PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys. Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end. Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized. Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope. Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.