Apoptosomes Structure and Procaspase Activation

描述（由申请人提供）：凋亡体结构和前半胱氨酸蛋白酶激活摘要程序性细胞死亡途径已经在真核生物中进化，以提供对发育线索、环境应激、细胞病原体和基因组损伤的适当反应。这些死亡途径也提供了一条防线，防止细胞不受限制地增殖。因此，细胞凋亡在维持人类健康，同时调节组织发育和稳态方面起着核心作用。 在这次更新中，我们将继续我们的联合结构和生物化学研究的染色体。这些平台由Apaf-1样蛋白组装而成，并激活内在细胞死亡途径中的半胱天冬酶原（pc）。Apaf-1卡和pc-9卡形成一个激活盘，位于轮状螺旋体的中心毂上方。pc-9中的盘和保守的接头在pc- 9催化结构域的激活中起重要作用，该催化结构域结合到中心枢纽。盘组装和结合pc-9催化结构域一起创建不对称蛋白水解机器。我们还发现，caspase-3通过一种新的反馈机制下调pc-9的活性，该机制使用了枢纽上的重叠结合位点。 在目标1中，将确定与pc-9和半胱天冬酶-3复合的人溶酶体的冷冻-EM结构。这些结构将为程序性细胞死亡的组装、激活和执行机制的生化研究提供框架。此外，结晶实验将针对调节β-螺旋桨和启动组装的细胞色素c之间的相互作用。在目标2中，将在近原子分辨率下确定果蝇染色体的结构。还将确定Dronc及其引发剂半胱氨酸蛋白酶原的暗小体的3D结构。这些数据将用于进行靶向诱变和生化实验，以了解Dark的功能。有趣的是，我们发现，几何形状的“活性位点”CARDs在人类，苍蝇和蠕虫的染色体的基态不同。在一个统一的假设，我们建议，在所有后生动物的启动proaspases将形成一个CARD-CARD盘，并被激活的邻近诱导协会的催化结构域与他们的染色体伴侣。当这些目标结合在一起时，应该揭示与人类健康和疾病密切相关的染色体功能的新方面。

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.