BaxΔ2 is an isoform of the proapoptotic Bcl-2 family member Bax that promotes cell death via caspase-8 activation. The C-terminus of BaxΔ2 has... Show moreBaxΔ2 is an isoform of the proapoptotic Bcl-2 family member Bax that promotes cell death via caspase-8 activation. The C-terminus of BaxΔ2 has been shown to be crucial for caspase-8 dependent cell death in colon cancer cells. However, it is unknown whether the C-terminal primary sequence or secondary structure is necessary for interaction with caspase-8. In this project, several BaxΔ2 C-terminal mutants were generated based on secondary structure predictions. Models showed that mutating Leu164 and Thr165 to Ala (LT-AA) would increase the probability of alpha helix formation, while mutating Leu164 to Pro (L-P) would decrease the probability of alpha helix formation. Expression of these mutant proteins in colon cancer HCT116 cells, showed that L164P, and not L164A/T165A, significantly impaired BaxΔ2 function. The mutant L164P proteins formed atypical aggregates, and their ability to induce cell death was also significantly decreased when compared to the wild type. These results indicate that the BaxΔ2 C-terminal tridimensional structure, and not the specific primary sequence, is critical for triggering aggregation-mediated cell death. M.S. in Biology, May 2018 Show less
