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。细胞凋亡异常是许多人类疾病的致病因素，包括癌症和缺血性心脑损伤。Bcl2和bax分别作为细胞凋亡的抑制者和促进者。任何一种蛋白的异常表达都会导致细胞凋亡的失调或发生凋亡性疾病。在正常的健康细胞中，bcl2锚定在线粒体外膜上，而bax则停留在胞浆中。在诱导细胞凋亡的过程中，Bcl2维持正常的膜功能，从而保护细胞器，从而保护细胞。相反，Bax插入线粒体膜并使其通透性。这两种蛋白质的结构都是在溶液中测定的，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上形成孔，如果它们形成了孔，它们的孔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的方法，并控制癌症和中风等疾病。我们的短期目标是确定为什么Bcl2抑制细胞凋亡，而结构相似的Bax促进它。我们的假设是，在BH3蛋白的作用下，Bcl2和Bax在线粒体膜上形成不同的结构，从而不同地调节线粒体膜的通透性和细胞凋亡。我们将解决三个具体目标。1)Bcl2与Bax和/或BH3-Only蛋白相互作用后膜上的结构是什么？2)Bcl2如何与Bax相互作用以阻止Bax形成细胞色素c释放孔？BH3-Only蛋白是否改变了Bcl2/Bax的相互作用？3)小分子化学Bcl2抑制剂如何抑制膜上的Bcl2？我们将产生一系列定点标记的、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。该项目的结果将为理解Bcl2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针对Bcl-2和Bax治疗癌症和心脏缺血等凋亡性疾病的治疗剂的先决条件。

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.