Abstract
We have developed an artificial liposome system that recapitulates intermembrane space protein release from mitochondria induced by Bcl-2 family proteins during apoptosis. Internally loaded fluorescein-dextrans are released in the same manner as in cytochrome c release from mitochondria. We have further characterized the system by using peptides from the BH3 region of pro-apoptotic Bcl-2 family proteins. The BH3 peptides from Bid and Bim activated Bax directly, while those from Bad and Bik showed no direct activation of Bax, but reversed the inhibition of Bcl-xL on Bax/Bid induced membrane permeabilization. These results are in good agreement with recently published observations obtained with mitochondria, supporting the hypothesis that our liposome system is physiological.We have also studied how p53, a tumor suppressor, could induce apoptosis in a transcription-independent manner using the liposome system. We observed that when p53 is over-expressed in the cytoplasm, it induces apoptosis by releasing cytochrome c from mitochondria. We then immunoaffinity purified p53 from UV-treated cells and added to liposomes in the presence of Bax, and it induced dextran release at a similar molar concentration range to that of Bid. We conclude that p53 can act like a BH3-only Bcl-2 family protein to directly activate Bax.The liposome system may be used to study how Bcl-2 family proteins as well as other proteins like p53 regulate mitochondrial outer membrane permeabilization in apoptosis. [Jpn J Physiol 54 Suppl:S28 (2004)]
