Structure-Function of Anti-Apoptosis Bcl-2 in Membranes

描述（由申请人提供）：细胞凋亡或程序性细胞死亡消除了后生动物中多余或受损的细胞。细胞凋亡异常参与了许多人类疾病的发病机制，包括癌症和缺血性心脏和脑损伤。Bcl-2和Bax分别作为细胞凋亡的抑制剂和促进剂发挥作用。任一蛋白质的异常表达导致细胞凋亡的失调或细胞凋亡性疾病的发作。Bcl- 2锚定在线粒体外膜上，而Bax则停留在正常健康细胞的胞质溶胶中。在凋亡诱导过程中，Bcl-2维持正常的膜功能，从而保护细胞器，从而保护细胞。Bax则插入线粒体膜并使其透化，这两种蛋白质的结构在溶液中被确定，并且彼此惊人地相似。在合成膜中，这两种蛋白质都形成孔，但我们不知道它们是否在线粒体膜中形成孔，如果它们形成孔，它们的孔如何不同地影响线粒体渗透性。我们的长期目标是找到干扰细胞凋亡程序和控制癌症和中风等疾病的方法。我们的短期目标是确定为什么Bcl-2抑制凋亡，而结构相似的Bax促进凋亡，我们的假设是Bcl-2和Bax在线粒体膜上形成不同的结构，从而差异调节膜通透性和凋亡。我们将讨论三个具体目标。1)Bcl-2与Bax和/或BH 3蛋白相互作用后，细胞膜中的Bcl-2结构是什么？2)Bcl-2如何与Bax相互作用以阻止Bax形成细胞色素c释放孔？BH 3-only蛋白改变Bcl-2/Bax相互作用吗？3)小的化学Bcl-2抑制剂如何抑制细胞膜中的Bcl-2？我们将产生一系列位点特异性标记的荧光或光反应性Bcl-2和Bax蛋白，并使用荧光和光交联方法来解决这些重要的细胞死亡或生存问题。该项目的结果将为理解Bcl-2和Bax在调节膜通透性和细胞凋亡中的功能奠定结构基础。这些知识是评估靶向Bcl-2和Bax的任何治疗剂用于治疗凋亡性疾病如癌症和心脏缺血的先决条件。

DESCRIPTION (provided by applicant): Apoptosis or programmed cell death eliminates redundant or damaged cells in metazoans. Abnormal apoptosis contributes to the pathogenesis of many human diseases including cancer and ischemic heart and brain injuries. Bcl-2 and Bax function as a suppressor and a promoter of apoptosis, respectively. Aberrant expression of either protein leads to dysregulation of apoptosis or onset of apoptotic diseases. Bcl- 2 is anchored on the mitochondrial outer membrane, while Bax stays in the cytosol in normal healthy cells. During apoptosis induction Bcl-2 maintains normal membrane function, thereby protecting the organelle and hence the cell. In contrast Bax inserts into the mitochondrial membrane and permeabilizes it. Structures of both proteins were determined in solution and are strikingly similar to each other. In synthetic membranes both proteins form pores, but we do not know whether they form pores in the mitochondrial membrane, and if they do, how their pores differentially affect the mitochondrial permeability. Our long-term goal is to find ways to interfere with the apoptosis program and control diseases such as cancer and stroke. Our short- term goal is to determine why Bcl-2 inhibits apoptosis, whereas the structurally similar Bax promotes it. Our hypothesis is that Bcl-2 and Bax form different structures in the mitochondrial membrane in response to BH3-only proteins, thereby differentially regulate the membrane permeability and apoptosis. We will address three specific aims. 1) What is the structure of Bcl-2 in membranes after it interacts with Bax and/or a BH3- only protein? 2) How does Bcl-2 interact with Bax to prevent Bax from forming a cytochrome c-releasing pore? Do BH3-only proteins change the Bcl-2/Bax interaction? 3) How do small chemical Bcl-2 inhibitors inhibit Bcl-2 in membranes? We will generate a series of site-specifically labeled, fluorescent or photoreactive Bcl-2 and Bax proteins, and use fluorescence and photocrosslinking approaches to resolve these important cell death-or-life issues. The results of the project will establish a structural basis for understanding the functions of Bcl-2 and Bax in regulating both membrane permeability and apoptosis. This knowledge is a prerequisite for evaluating any therapeutic agents that target Bcl-2 and Bax for treatment of apoptotic diseases like cancer and cardiac ischemia.