Bax is one of the two key proteins (Bax and Bak) of the Bcl-2 family that control the mitochondrial pathway of apoptosis. In healthy cells, Bax locates in the cytosol and is maintained in the inactive state. The C-termina! transmembrane (TM) domain of Bax, which is responsible for mitochondria targeting, is folded into a hydrophobic pocket on the Bax protein. Apoptotic signals induce conformational changes of the Bax protein and releases the TM domain from the pocket. The activated form of Bax protein migrates to mitochondria. [After insertion into mitochondrial outer membrane, Bax induces the release of pro-apoptotic proteins from mitochondria (cytochrome c, Smac, AIF, etc.) and disruption of mitochondrial membrane potential. These events lead to activation of caspases and apoptotic cell death. The mechanism that maintains Bax in its inactive state in healthy cells is not known until recently we and another group have identified the proteins (Humanin and other proteins) that bind to and suppress Bax activation. However, the mechanism by which apoptotic stimuli release Humanin from Bax and activate Bax remains unknown. We hypothesize that apoptotic stimuli activate unknown protein factor(s) that binds to or modify Bax-Humanin complex, causing the release of Humanin and the activation of Bax. Consequently, the specific aims of this proposal are: 1) To identify the protein factor(s) that activates Bax in response to apoptotic stimuli; 2)To investigate the physiological function of the identified Bax activating factor(s) in apoptosis. The Bax activating proteins will be identified by biochemical fractionation of cytosolic preparation from apoptotic cells. The physiological function of the identified Bax activating proteins will be investigated by overexpression in cancer cells through transfection and by knocking-down their expression using siRNA technique. These studies will help improve our knowledge on the mechanism of Bax activation, a critical decision-making process in apoptosis.

R21CA111765

2005-09-01

2008-08-31