Studies on the role of Bid and Mcl-1 in the regulation of apoptosis
The Bcl-2 family of proteins are critical regulators of mitochondrial function and deregulation during apoptosis. To this effect, the 'BH3-only' members of this family play an important role in initiating many of the apoptotic signalling pathways. This thesis focuses on the BH3-only protein Bid. We have used the active form of Bid, tBid, as bait in a yeast two-hybrid assay to identify novel interaction partners for this protein. In combination with a GST pull-down assay we identified two novel interaction partners for tBid. The proteins identified were Mcl-1 and EID-1. Mcl-1 is an anti-apoptotic Bcl-2 family member, although its role in apoptosis has not been as extensively studied as other anti-apoptotic proteins such as Bcl-2 and Bcl-XL. EID-1 was first identified as a pRb interacting protein that has been shown to inhibit differentiation of myoblasts. However, there is no data to suggest that EID-1 may have a role in apoptosis, and to date all data suggests that the protein has a nuclear function rather than a cytoplasmic one. During the course of this work, we observed that Mcl-1 is cleaved during apoptosis of Jurkat T cells. We have characterised this cleavage and found it to be as a result of caspase cleavage. There are two caspase cleavage sites in Mcl-1. These occur at Asp127 and Asp157. Many Bcl-2 family members are cleaved by caspases during apoptosis. In particular, anti-apoptotic family members cleaved by caspases are converted into pro-apoptotic proteins. In contrast to this we did not observe any pro- apoptotic activity associated with the cleavage fragments. We also found that the PEST region in Mcl-1 did not regulate its half-life in FDCP-1 cells, confirming results already observed in U937 cells. While further work needs to be carried out, the results presented here provide a number of important observations that may help to further the understanding of how both Bid and Mcl-1 contribute to apoptosis.