Structural Biology of Apoptosomes and Related Signaling Complexes

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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.