Mechanisms of Bax Activation in Apoptosis

描述(申请人提供)：Bax是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位于胞浆中，并维持在不活跃状态。C-终结者！Bax的跨膜(TM)结构域负责线粒体的靶向，被折叠到Bax蛋白上的疏水口袋中。凋亡信号诱导Bax蛋白的构象变化，并从口袋释放TM结构域。Bax蛋白的活化形式迁移到线粒体。[Bax插入线粒体外膜后，可诱导线粒体释放促凋亡蛋白(细胞色素c、Smac、AIF等)。)和线粒体膜电位的破坏。这些事件导致半胱氨酸天冬氨酸酶的激活和细胞的凋亡。在健康细胞中维持Bax处于不活跃状态的机制尚不清楚，直到最近我们和另一个小组已经确定了与Bax结合并抑制Bax激活的蛋白质(人素和其他蛋白质)。然而，凋亡刺激从Bax释放人蛋白并激活Bax的机制仍不清楚。我们假设凋亡刺激激活未知的蛋白因子(S)，该因子结合或修饰bax-人蛋白复合体，导致人蛋白的释放和bax的激活。因此，本提案的具体目的是：1)鉴定在细胞凋亡刺激下激活bax的蛋白因子(S)；2)研究已鉴定的bax激活因子(S)在细胞凋亡中的生理功能。Bax激活蛋白将通过对凋亡细胞的胞浆制备进行生化分级来鉴定。已鉴定的Bax激活蛋白的生理功能将通过转染法在癌细胞中过度表达以及通过siRNA技术下调其表达来研究。这些研究将有助于提高我们对Bax激活机制的了解，Bax激活是细胞凋亡中的一个关键决策过程。

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.