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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM063834-09A1
Application #
8299718
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Maas, Stefan
Project Start
2001-07-01
Project End
2015-12-31
Budget Start
2012-04-01
Budget End
2012-12-31
Support Year
9
Fiscal Year
2012
Total Cost
$298,813
Indirect Cost
$108,813
Name
Boston University
Department
Physiology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Yuan, Shujun; Akey, Christopher W (2013) Apoptosome structure, assembly, and procaspase activation. Structure 21:501-15
Yuan, Shujun; Topf, Maya; Reubold, Thomas F et al. (2013) Changes in Apaf-1 conformation that drive apoptosome assembly. Biochemistry 52:2319-27
Yuan, Shujun; Yu, Xinchao; Asara, John M et al. (2011) The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3. Structure 19:1084-96
Yuan, Shujun; Yu, Xinchao; Topf, Maya et al. (2011) Structure of the Drosophila apoptosome at 6.9ýýa resolution. Structure 19:128-40
Yuan, Shujun; Yu, Xinchao; Topf, Maya et al. (2010) Structure of an apoptosome-procaspase-9 CARD complex. Structure 18:571-83