Study Of The Interaction Between BclGs And JAB1 And Its Role In The Regulation Of Apoptosis

The Bcl-2 family members are the key regulators of cell apoptosis at the mitochondria level. The BH3-only pro-apoptotic member BclGs(Bcl-Gonad short form) was unique among the family due to its highly specific expression in human testis and has been demonstrated to induce apoptosis dependent of the BH3 domain. However, the mechanism of BclGs induced apoptosis is still unclear. In this study, we found the role of BclGs induced apoptosis. Moreover, we identified JAB1 as a novel BclGs-specific binding protein through a yeast two-hybrid screening in a human testis cDNA library. Furthermore, our study elucidated the interaction between BclGs and JAB1 and its role in the regulation of apoptosis.(1) HeLa cells were transfected with BclGs. The expression level of Bax in total cell lysate detected by Western blot. Bax expression was upregulated in cells when BclGs was overexpressed. Moreover, Bax translocation from cytosol and mitochondria was also analyzed. Cytosolic and mitochondrial proteins were extracted from HeLa cells transfected with BclGs and Bax and cytochrome c expression was analyzed by western blot. It was shown that Bax level increased in mitochondria and decreased in cytosol fractions in cells transfected with BclGs. Accordingly; we examined the distribution of cytochrome c in cytosol and mitochondria. The release of cytochrome c from mitochondria into the cytosol also occurred in response to BclGs overexpression. In order to investigate whether BclGs-induced cell apoptosis activated caspases. Caspase-3 activation in HeLa cells was analyzed by western blot. Cleavage of caspase-3 was clearly observed in HeLa cells transfected with BclGs. Thegeneral caspase inhibitor z-VAD-FMK could abrogate BclGs-induced apoptosis.(2) Moreover, we identified JAB1 as a novel BclGs-specific binding protein through a yeast two-hybrid screening in a human testis cDNA library. An analysis of BclGs deletion constructs showed that N-terminal deleted BclGs mutants could not interact with JAB1, whereas C-terminal deleted BclGs mutants could. Our study showed that N-terminal 1-67 amino acids of BclGs were required for this interaction. We also demonstrated that BclGs was mainly localized in the cytoplasm, whereas JAB1 mainly in the cytoplasm and partially in the nucleus when overexpressed alone, and that BclGs and JAB1 were colocalized in the cytoplasm when co-expressed.