REGULATION OF APOPTOSIS BY BH3 DOMAINS IN A CELL-FREE SYSTEM

Background: The Bcl-2 family of proteins plays a key role in the regul ation of apoptosis, Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis, Competitive dim erisation between family members is thought to regulate their function , Homologous domains within individual proteins are necessary for inte ractions with other family members and for activity, although the spec ific mechanisms might differ between the pro-apoptotic and anti-apopto tic proteins. Results: Using a cell-free system based on extracts of X enopus eggs, we have investigated the role of the Bcl-2 homology domai n 3 (BH3) from different members of the Bcl-2 family, BH3 domains from the pro-apoptotic proteins Bar and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as de termined by the rapid activation of specific apoptotic proteases (casp ases) and by DNA fragmentation, The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytopla sm, involves the release of cytochrome c from mitochondria and is anta gonistic to Bcl-2 function, Short peptides, corresponding to the minim al sequence of BH3 domains required to bind anti-apoptotic Bcl-2 famil y proteins, also trigger apoptosis in this system, Conclusions: The BH 3 domains of pro-apoptotic proteins are sufficient to trigger cytochro me c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis, The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides ma y provide the basis for engineering reagents to control the initiation of apoptosis.