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后生动物中多余或受损的细胞。细胞凋亡异常与癌症、缺血性心脑损伤等多种人类疾病的发病有关。Bcl-2和Bax分别是细胞凋亡的抑制因子和启动因子。任何一种蛋白的异常表达都会导致细胞凋亡的失调或凋亡性疾病的发生。在正常健康细胞中，Bcl- 2锚定在线粒体外膜上，而Bax则停留在细胞质中。在细胞凋亡诱导过程中，Bcl-2维持正常的膜功能，从而保护细胞器和细胞。相反，Bax插入线粒体膜并使其通透。这两种蛋白质的结构都是在溶液中确定的，它们彼此惊人地相似。在合成膜中，这两种蛋白质都形成孔洞，但我们不知道它们是否在线粒体膜上形成孔洞，如果是，它们的孔洞如何不同地影响线粒体的通透性。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抑制细胞凋亡，而结构相似的Bax促进细胞凋亡。我们的假设是Bcl-2和Bax响应BH3-only蛋白在线粒体膜上形成不同的结构，从而不同地调控膜通透性和细胞凋亡。我们将实现三个具体目标。1) Bcl-2与Bax和/或仅BH3蛋白相互作用后，膜中的结构是什么？2) Bcl-2如何与Bax相互作用，阻止Bax形成细胞色素c释放孔？仅bh3蛋白会改变Bcl-2/Bax的相互作用吗？3)小化学Bcl-2抑制剂如何抑制膜中的Bcl-2 ？我们将生成一系列位点特异性标记，荧光或光反应的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命问题。该项目的研究结果将为了解Bcl-2和Bax在调节细胞膜通透性和细胞凋亡中的功能奠定结构基础。这一知识是评估任何靶向Bcl-2和Bax治疗凋亡性疾病（如癌症和心脏缺血）的治疗药物的先决条件。

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.