| BRE (B&barbelow;rain and R&barbelow;eproductive E&barbelow;xpressed) was first identified as a ubiquitously expressed gene whose transcription could be down-regulated by DNA damaging agents. Subsequently in a yeast two-hybrid screen of a mouse cDNA library using the cytoplasmic region of TNF-RI as bait, BRE was identified as one of the binding proteins. Overexpression of this protein in transfected cell lines down-regulated NF-kappaB activation induced by TNF-alpha. In this thesis, I report my work on further characterization of this protein. The complete BRE cDNA gene sequences of mouse, and non-human primates have been sequenced by us. These sequences in conjunction with those found in the GeneBank reveal an evolutionarily highly conserved protein sequence of BRE. The human and mouse protein differ only at 3 residues in the entire sequence of 383 amino acids. Both species, however, generate different sets of multiple transcript isoforms through different modes of alternative splicing. My work only focused on the protein product of the main transcript.; I found that BRE is localized in cytosol and nucleus, but not mitochondria. It may be post-translationally modified to result in an increase of around 4∼5kDa. This modification, which is not ubiquitination, sumoylation or phosphorylation, depends on the intact N- but not the C-terminus. However, TNF-alpha or Fas stimulation promotes interaction of BRE with ubiquitinated, sumoylated or phosphorylated proteins. By overexpression of human BRE in transfected human and mouse cell lines, I observed that these cell lines showed significantly reduced apoptosis to TNF-alpha in the presence of cycloheximide. Furthermore, BRE overexpression inhibits apoptosis triggered by cycloheximide, Fas, FADD, mitochondria-stress stimuli. Analysis of the apoptotic events affected by BRE overexpression revealed a novel anti-apoptotic mechanism that involves inhibiting the cross-talk between the death receptor-mediated and mitochondria-dependent apoptotic pathway. Transfer of the mouse Lewis lung carcinoma 3LL-D122 subclones overexpressing human BRE into the host C57BL/6 and the immunodeficient nude mice resulted in accelerated tumor growth compared with the untransfected and empty vector-transfected D122. As the transfected D122 cells did not grow faster in cell culture condition, I conclude that the enhancement of in vivo tumor growth by BRE overexpression was due to better tumor survival in vivo, possibly through reduced sensitivity to TNF-alpha- and Fas-mediated apoptosis induced by the anti-tumor immunosurveillance. Using co-immunoprecipitation, I confirmed that BRE can indeed bind to the endogenous TNF-RI. Furthermore, I found binding of BRE to the endogenous Fas as well, which is consistent with my above observation showing down-modulation of Fas-mediated apoptosis by BRE. BRE dissociates rapidly from TNF-RI, but remains associated with Fas, upon activation of the respective death receptor, probably reflecting the difference between the two receptors in forming the death-inducing signaling complexes, with which BRE may interact. Taken together, these data indicate that BRE is an anti-apoptotic protein that down-modulates the pro-apoptotic signaling of TNF-RI and Fas by inhibiting the activation of the mitochondrial apoptotic events. |
