Structural Biology of Apoptosomes and Related Signaling Complexes

程序性细胞死亡(细胞凋亡)是一个个体细胞被终止以使有机体受益的过程。然而，许多癌症抑制凋亡途径以允许细胞增殖，而退行性疾病可能上调凋亡以过早杀死细胞。在固有的细胞死亡途径中，细胞色素c从线粒体中释放出来，并在dATP存在的情况下与APAF-1相互作用。这触发了凋亡体组装，由此产生的平台招募并激活了proaspase-9。然后，这种全酶激活刽子手的蛋白水解酶，杀死细胞。值得注意的是，APAF-1组装最近被认为可能是导致严重抑郁症综合征(MDD)的一个因素。在果蝇中，一个类似的细胞杀伤机是由Dark形成的，它是一种与APAF-1相关的杀手，它顺序地激活原天冬氨酸酶Done和Drice。 在特定的目标1和2中，我们将确定APAF-1和Dark凋亡体的高分辨率三维结构。此外，还将从它们的顶端原天冬氨酸酶中获得带有前域的“全酶”结构。在这些研究中，将使用电子冷冻显微镜和单粒子方法、X射线结晶学和同源建模来创建原子模型。这些研究将提供人类和果蝇凋亡体的详细图景，还将揭示前天冬氨酸酶结合时发生的构象变化。因此，特定的目标1和2将使我们更清楚地了解这些大平台如何在内在的细胞死亡途径中组装和发挥作用。 在促炎途径中，APAF-1相关蛋白NOD2感知细菌并组装一个信号平台。通过一系列步骤，该复合体激活转录因子NFkB，该转录因子上调涉及先天免疫或获得性免疫的基因。NOD2的缺陷与慢性小肠炎(克罗恩病)和布劳综合征有关。在具体目标3中，将组装一个NOD2信号平台，并确定该复合体的第一个三维结构。从长远来看，这些研究将帮助我们了解NOD2如何在对胃肠道细菌的先天免疫反应中发挥作用。

Programmed cell death (apoptosis) is a process whereby individual cells are terminated to benefit the organism. However, many cancers inhibit apoptotic pathways to allow cell proliferation, while degenerative diseases may up-regulate apoptosis to kill cells prematurely. In the intrinsic cell death pathway, cytochrome c is released from mitochondria and interacts with Apaf-1 in the presence of dATP. This triggers apoptosome assembly and the resulting platform recruits and activates procaspase-9. This holo-enzyme then activates executioner procaspases which kill the cell. Significantly, Apaf-1 assembly has recently been implicated as a possible contributing factor to major depression syndrome (MDD). In Drosophila, a similar cell killing machine is formed by Dark, an Apaf-1 Related Killer which sequentially activates the procaspases Drone and DrICE. In Specific Aims 1 and 2, we will determine high resolution, 3-dimensional structures of the Apaf-1 and Dark apoptosomes. In addition, structures will be obtained of "holo-enzymes" with prodomains from their apical procaspases. In these studies, electron cryo-microscopy and single particle methods, X-ray crystallography and homology modeling will be used to create atomic models. These studies will provide a detailed picture of human and Drosophila apoptosomes and will also reveal conformational changes that occur when procaspases bind. Thus, Specific Aims 1 and 2 will give a clearer understanding of how these large platforms assemble and function in the intrinsic cell death pathway. In pro-inflammatory pathways, the Apaf-1 related protein NOD2 senses bacteria and assembles a signaling platform. Through a series of steps, this complex activates the transcription factor NFkB, which up-regulates genes involved in innate or adaptive immunity. Defects in NOD2 are associated with chronic inflammation of the small intestine (Crohn's disease) and with Blau's syndrome. In Specific Aim 3, a NOD2 signaling platform will be assembled and the first 3-dimensional structure of this complex will be determined. In the long term, these studies will help us to understand how NOD2 functions in the innate immune response to bacteria in the gastro-intestinal tract.