Medical Biology - Theses
Permanent URI for this collection
Search Results
1 - 1 of 1
-
ItemThe Bcl-2 apoptotic switch – clarifying the functions of proapoptotic and prosurvival players( 2015)The Bcl-2 apoptotic switch is the key decision point in the intrinsic pathway of apoptosis. The study of Bcl-2 family protein function in vitro has been hampered by the lack of full-length recombinant BH3-only proteins, the ‘stimuli’ of the Bcl-2 family. Therefore, a panel of chimeras of Bid with the BH3 domains of other BH3-only proteins was generated to dissect the layers of regulation governing the Bcl-2 effector proteins Bak and Bax. Using Bid BH3 chimeras and BH3 peptides on isolated mitochondria and liposomes, we concluded that most BH3-only proteins could directly activate the effector Bcl-2 family proteins Bak and Bax. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Moreover, Bak and Bax were activated by the same stimuli, in contrast to a recent report. The Bid chimeras were also used to investigate a mechanism of prosurvival Bcl-2 protein function in which a prosurvival protein causes resistance to apoptosis by binding activated Bak (MODE 2) rather than BH3-only proteins (MODE 1). The findings were validated using full-length proteins on isolated mitochondria and in cultured cells. In response to BH3-only proteins, the prosurvival proteins Mcl-1 and Bcl-xL were able to prevent cytochrome c release or apoptosis via MODE 2. In particular, MODE 2 interactions between Bak and Mcl-1 caused profound resistance to Bid. The studies presented here are the first direct evidence that MODE 2 can protect cultured cells without over-expressed prosurvival proteins, and