APOPTOSIS INHIBITOR 5 (Api5) ACETYLATION: ROLE IN APOPTOSIS

Abstract

Apoptosis Inhibitor 5, Api5 is a nuclear protein which inhibits apoptosis upon growth factor deprival and is up regulated in various cancers. Api5 levels have been shown to be cell cycle regulated. Role of Api5 in cellular processes is not completely understood, especially it‟s regulation by post translational modifications is least explored. Api5 lysine-251 acetylation, only post translational modification known to occur on Api5, was shown to negatively regulate apoptosis induced by serum starvation. Here, we tried to understand the mechanism through which acetylation regulates DNA damage induced apoptosis. In the process of standardizing dose regimen for the apoptotic assays, we observed that varying doses of camptothecin treatment did not induce DNA fragmentation. However, UV damage induced time dependent activation of caspase-9 and PARP-1 in MCF-7 cells. Further, it was observed that UV damage also induced pre-apoptotic nuclear morphology and cytoplasmic localization of lamin B1. Previous studies in our lab demonstrated that Api5 interacts with TopBP1, a mediator protein in single stranded break induced DNA damage. Further, using deletion constructs it was showed that lysine 251 lies in that region of Api5 which interacts with TopBP1. Therefore, we also tried to investigate its role in interaction with TopBP1 in vivo. Towards this end, we have successfully generated siRNA resistant HA-Api5 and Api5-mVenusC1 acetylation mutants (an uncharged mutant, acetylation-deficient and a constitutive acetylation mimic). Also, our preliminary experiments suggested that Api5 is indispensible for cell survival. Additionally, this study also focused on the degradation pathway of Api5. We observed that Api5 levels were not altered by MG-132 (proteasome inhibitor) treatment, suggesting that Api5 may not be undergoing degradation through proteosomal pathway. In summary, with further investigation, this study could potentially give new insights in understanding the role of posttranslational modifications on Api5 in carrying out its function.