Mechanisms of Bax Activation in Apoptosis

描述（申请人提供）：Bax是Bcl-2家族中控制线粒体凋亡途径的两个关键蛋白（Bax和Bak）之一。在健康细胞中，Bax位于细胞质中并保持无活性状态。C-termina !Bax的跨膜（TM）结构域负责线粒体靶向，折叠成Bax蛋白上的疏水口袋。凋亡信号诱导Bax蛋白的构象改变，并从口袋中释放TM结构域。活化形式的Bax蛋白迁移到线粒体。[插入线粒体外膜后，Bax诱导线粒体释放促凋亡蛋白（细胞色素c、Smac、AIF等），破坏线粒体膜电位。]这些事件导致半胱天冬酶的激活和凋亡细胞死亡。直到最近，我们和另一个研究小组发现了结合并抑制Bax激活的蛋白质（Humanin和其他蛋白质），才知道在健康细胞中维持Bax处于失活状态的机制。然而，凋亡刺激从Bax中释放Humanin并激活Bax的机制尚不清楚。我们假设凋亡刺激激活未知的结合或修饰Bax-Humanin复合物的蛋白因子，导致Humanin的释放和Bax的激活。因此，本提案的具体目的是：1)确定在凋亡刺激下激活Bax的蛋白因子；2)研究鉴定的Bax激活因子在细胞凋亡中的生理功能。Bax激活蛋白将通过凋亡细胞的细胞质制备的生化分离来鉴定。我们将通过转染和siRNA技术敲低Bax激活蛋白在癌细胞中的过表达来研究其生理功能。这些研究将有助于提高我们对Bax活化机制的认识，Bax活化是细胞凋亡的关键决策过程。

DESCRIPTION (provided by applicant): 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.