Studies of germinal center B cell survival

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DESCRIPTION (provided by applicant): Coordinate balance between cell survival and apoptosis is crucial for development and homeostasis. While most forms of apoptosis occur through the activation of caspases, evidence of apoptosis inducing factor (AIF)-mediated caspase-independent pathways has emerged. However, the mechanisms that rescue cells from caspase-independent apoptosis have not been determined. This study focuses on PRELI, a mitochondrial protein that possesses tandem repeats of the LEA (late embryogenesis abundant) motif, which can abrogate apoptosis induced by staurosporine, TNF-a and UV irradiation. PRELI can maintain the integrity of mitochondria, prevent cytochrome c release and protect cells from caspase-dependent and independent apoptosis. Our data also show that PRELI prevents AIF translocation from the mitochondria to the nucleus and suppress DMA fragmentation. The relevance of PRELI as a suppressor of apoptosis was substantiated by two targeted perturbations of its function: (i) deletion of its functional LEA motif and (ii) siRNA silencing in human cells. The present study thus reveals an evolutionarily conserved mechanism that concomitantly opposes caspase-dependent and independent apoptosis. The specific aims are: I. To assess the role of PRELI in the control of apoptosis. We shall test the effect of PRELI on the respiratory chain, reactive oxygen species (ROS) production and Ca2+ fluxes by measurements of enzyme activity and the use of fluorescent ion-binding probes. To investigate putative interactions of PRELI with other proteins, we shall conduct protein-profiling screens, yeast two-hybrid analysis, tagged-protein pull-downs, kinase assays, and western blottings; II. To assess in vivo the effects of PRELI gene overexpression and inactivation. We shall generate two distinct mouse models: (a) Transgenic mice overexpressing PRELI under the control of the f enhancer (Ef) will be produced to investigate whether PRELI-mediated survival leads to enhanced lymphocyte expansion and proliferation, (b) Tissue-specific PRELI-deficient mice will be generated to investigate the consequences of the inactivation of its gene on B lymphocyte survival during their maturation and differentiation. The B cell-targeted deletion will be obtained by breeding mice carrying the floxed PRELI gene with mice expressing CD19-driven Cre-recombinase.