Apoptosomes Structure and Procaspase Activation

在真核生物中，程序性细胞死亡途径已经进化为对发育线索、环境胁迫、细胞病原体和基因组损伤提供适当的反应。这些死亡途径也为不受限制的细胞增殖提供了一道防线。因此，细胞凋亡在维持人体健康中起着核心作用，同时调节组织发育和体内平衡。在这次更新中，我们将继续我们的细胞凋亡的结构和生化结合研究。这些平台由Apaf-1样蛋白组装并激活内在细胞死亡途径中的原aspase （pc）。Apaf-1卡和pc-9卡形成一个激活盘，位于轮状凋亡细胞的中央枢纽上方。磁盘和pc-9中的保守连接体在pc-9催化结构域的激活中起重要作用，这些催化结构域与中心集线器结合。磁盘组装和结合的pc-9催化结构域一起创造了一个不对称的蛋白质水解机器。我们还发现caspase-3通过一种新颖的反馈机制下调pc-9的活性，该机制使用中枢上的重叠结合位点。在Aim 1中，冷冻电镜结构将确定与pc-9和caspase-3复合物的人凋亡细胞。这些结构将为程序性细胞死亡中潜在的组装、激活和执行机制的生化研究提供框架。此外，结晶实验将针对调控β -螺旋桨和启动组装的细胞色素c之间的相互作用。在Aim 2中，果蝇凋亡细胞的结构将在近原子分辨率下确定。暗细胞凋亡的三维结构与Dronc，其引发剂procaspase也将被确定。这些数据将用于开展靶向诱变和生化实验，以了解Dark的功能。有趣的是，我们发现在人类、苍蝇和蠕虫细胞凋亡的基态中，“活性位点”卡的几何形状是不同的。在一个统一的假设中，我们提出所有后生动物的启动原胞浆酶将形成一个CARD-CARD磁盘，并通过邻近诱导的催化结构域与其凋亡伙伴的关联而被激活。当这些目标结合在一起时，应该揭示与人类健康和疾病相关的凋亡功能的新方面。

DESCRIPTION (provided by applicant): Apoptosome Structure and Procaspase Activation Abstract Programmed cell death pathways have evolved in eukaryotes to provide appropriate responses to developmental cues, environmental stress, cellular pathogens and genomic insults. These death pathways also provide a line of defense against unrestricted cell proliferation. Hence, apoptosis plays a central role in the maintenance of human health, while regulating tissue development and homeostasis. In this renewal, we will continue our combined structural and biochemical studies of apoptosomes. These platforms assemble from Apaf-1 like proteins and activate procaspases (pc) in the intrinsic cell death pathway. Apaf-1 CARDs and pc-9 CARDs form an activation disk that sits above the central hub of the wheel-like apoptosome. The disk and a conserved linker in pc-9, play important roles in the activation of pc- 9 catalytic domains, which bind to the central hub. Together, disk assembly and bound pc-9 catalytic domains create an asymmetric proteolysis machine. We have also shown that caspase-3 down-regulates pc-9 activity through a novel feedback mechanism which uses overlapping binding sites on the hub. In Aim 1, cryo-EM structures will be determined of the human apoptosome in complexes with pc-9 and caspase-3. These structures will provide a framework for biochemical studies on the mechanisms underlying assembly, activation and execution in programmed cell death. In addition, crystallization experiments will target interactions between regulatory beta-propellers and cytochrome c that kick-start assembly. In Aim 2, a structure of the Drosophila apoptosome will be determined at near atomic resolution. A 3D structure of the Dark apoptosome with Dronc, its initiator procaspase will also be determined. This data will be used to carry out targeted mutagenesis and biochemical experiments to understand the function of Dark. Intriguingly, we find that the geometry of "active site" CARDs differs in the ground states of human, fly and worm apoptosomes. In a unifying hypothesis, we propose that initiator procaspases in all metazoans will form a CARD-CARD disk and be activated by proximity induced association of catalytic domains with their apoptosome partners. When taken together, these Aims should reveal new aspects of apoptosome function that are germane to human health and disease. PUBLIC HEALTH RELEVANCE: Apoptosomes assemble in the cytoplasm when cells are subjected to severe environmental or pathogenic stress. These complexes bind and activate proteases that subsequently kill the cell from the inside out. Under these conditions, cell contents are recycled by adjacent cells to benefit the organism. This program of tightly controlled cell death also plays an important role in development of the brain and other organs. In addition, the intrinsic pathway also helps to maintain cellular homeostasis in various tissues. Mutations in programmed cell death pathway components are required to convert many cells to cancers, along with mutations that up-regulate pro-growth genes. Hence, programmed cell death is a first line of defense against many cancers.