Variant of Genetic Suppressor Element (GSE) approach, based on replication competent avian retroviral system, reveals novel mechanism of FAS-induced apoptosis that involves mitochondrial cytochrome b

New modification of Genetic Suppressor Element (GSE) selection method for identification of novel genes involved in regulation of FAS apoptosis is described in this work. GSE library in replication-competent avian retroviral vector RCASBP(A) was introduced into chicken cells carrying human FAS receptor. These cells were subjected to FAS apoptosis by treatment with FAS agonistic antibody. Replication competency of RCASBP vector allowed for several rounds of screening by efficient rescue of viral particles carrying GSEs inactivating particular events in FAS-dependent apoptosis for subsequent selections. We were able to isolate several GSEs inhibiting FAS-dependent apoptosis, and these GSEs were individually confirmed in FAS survival assays in chicken and human systems, where they demonstrated substantial protection from FAS apoptosis.; Two GSEs and their corresponding genes were characterized in detail. The effects GRPE (HSP 70 ATPase) and Moesin (cytoskeletal protein involved in regulation of cellular receptors) in FAS apoptosis were dissected by employing short hairpin RNA (shRNA) approach. shRNA directed against GRPE protein had a protective effect against FAS apoptosis. We concluded that GRPE expression is required for propagation of FAS apoptosis and its corresponding GSE acts through dominant-negative mechanism. In accordance with this result, full-length GRPE over-expression was found to be toxic to the cells. shRNA directed against Moesin protein did not have any effect against FAS apoptosis. However, full-length Moesin over-expression exerted the anti-apoptotic effect in the cells. Thus, its corresponding GSE presumably acts through the minimal functional domain mechanism.; There were four GSEs isolated which encode for the fragments of different subunits of mitochondrial electron-transport chain. All of these GSEs correspond to mitochondria-encoded key subunits of this complex. We decided to concentrate our studies on one of the GSEs which corresponds to Cytochrome b (Cyt b) subunit of complex III. We came up with the hypothesis that release of particular components of mitochondria, such as Cyt b, in addition to cytochrome c is required for propagation of FAS apoptosis. The involvement of mitochondrial Cyt b in regulation of FAS apoptosis was investigated in detail. From the results of the immunostaining and fractionation experiments it can be seen that induction of apoptosis induces rapid destabilization, cleavage and relocalization of C-terminal fragment of Cyt b from mitochondria to cytoplasm during the course of FAS apoptosis. The action of the full-length and C-terminal fragment of Cyt b in cytoplasm was determined. Due to the differences in the nuclear and mitochondrial code, the sequence of the full-length Cyt b had to be corrected for cytoplasmic expression. From the results of these experiments, we can judge that cytoplasmic targeting of mitochondrial Cyt b protein induces apoptosis, thus contributing to regulation of FAS apoptosis. Potential mediators of Cyt b apoptotic function in cytoplasm were identified and confirmed. Major Vault Protein (MVP) was isolated as the protein interacting with Cyt b in cytoplasm. Our data let us establish a link between FAS apoptosis and vault complex through cytoplasmic release of Cyt b.